A first trial to supplement local hardwood sawdust at the first soaking for the cultivation of Shiitake (Lentinula edodes)

Experimental design and treatment

The experiment was arranged in a completely randomized design with six treatments and six replicates per treatment (six bags of one kg per treatment). Initially, each replicate contained 800 g of sawdust and 200 g of wheat bran. Sawdust was supplemented or not with nano-supplement and treatments were as follows: T1: OS-WB, T2: OS-WB+ nano-amino, T3: MS-WB, T4: MS-WB+ nano-amino, T5: AS-WB, and T6: AS-WB+ nano-amino.

Evaluation of sawdust properties

Various analytical tests were performed to assess the physico-chemical properties of each sawdust type at the Lebanese Agricultural Research Institute (LARI)-Tal Amara station using a sample of 0.5 kg. Sawdust pH was evaluated on filtrates of sawdust samples using a pH-meter (ADWA AD 132). Crude protein was assessed using the Micro-Kjeldahl method (N × 6.25) (Association of Official Chemistry (AOAC), 1995). Moisture content determination followed (Association of Official Chemistry (AOAC), 2005a): two grams of substrate samples were dried under pressure of 13332.2 Pa to a constant weight at 95–100 °C. Ash content was determined according to the Association of Official Chemistry (AOAC) (2005a): two grams of sample were weighed into a porcelain crucible and preheated to 600 °C for 2 h in a temperature-controlled furnace. Then, the crucible was transferred to a desiccator, cooled, and weighed, and the percentage of ash was recorded as follows:

$$Ash\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\left(W_1\right)\left(\mathrm{g}\right)-\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{h}\mathrm{d}\mathrm{r}\mathrm{y}\mathrm{i}\mathrm{n}\mathrm{g}\left(W_2\right)\left(\mathrm{g}\right)}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\left(W_1\right)\left(\mathrm{g}\right)}}$$

Crude fat was assessed by acid hydrolysis method according to the Association of Official Chemistry (AOAC) (2005b). Dry matter, organic matter, carbon, nitrogen, and carbohydrates contents of sawdust were determined by calculation as follows:

$$\mathrm{D}\mathrm{r}\mathrm{y}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\left(\mathrm{\%}\right)=100-\mathrm{m}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\left(\mathrm{\%}\right)$$

$$\mathrm{O}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\left(\mathrm{\%}\right)=100-\mathrm{a}\mathrm{s}\mathrm{h}\left(\mathrm{\%}\right)$$

$$\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\left(\mathrm{\%}\right)}{1.72}}$$

$$\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\left(\mathrm{\%}\right)=\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\left(\mathrm{\%}\right)\mathrm{x}6.25$$

$$\begin{array}{rl}\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{s}\left(\mathrm{\%}\right)=& 100-\mathrm{m}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\left(\mathrm{\%}\right)-\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\left(\mathrm{\%}\right)\\ & -\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{f}\mathrm{a}\mathrm{t}\left(\mathrm{\%}\right)-\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{s}\left(\mathrm{\%}\right)\end{array}$$

Crude fibers content was assessed based on the Weende method according to the Association of Official Chemistry (AOAC) (2005c). Fiber fractions (acid detergent fiber: ADF, neutral detergent fiber: NDF, and acid detergent lignin: ADL) were measured by the Fibertherm methodology according to Wannasawang et al. (2023). Consequently, the contents of cellulose, hemicellulose, and lignin were calculated as follows:

$$\mathrm{L}\mathrm{i}\mathrm{g}\mathrm{n}\mathrm{i}\mathrm{n}\left(\mathrm{\%}\right)=\mathrm{A}\mathrm{D}\mathrm{F}\left(\mathrm{\%}\right)$$

$$\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e}\left(\mathrm{\%}\right)=\mathrm{N}\mathrm{D}\mathrm{F}\left(\mathrm{\%}\right)-\mathrm{A}\mathrm{D}\mathrm{F}\left(\mathrm{\%}\right)$$

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e}\left(\mathrm{\%}\right)=\mathrm{A}\mathrm{D}\mathrm{F}\left(\mathrm{\%}\right)-\mathrm{A}\mathrm{D}\mathrm{L}\left(\mathrm{\%}\right)$$

Substrates preparation

A Pro Wood chipper was used to reduce apple wood logs sourced from a local organic apple orchard to sawdust size. Oak and maple sawdust sourced from ‘Mushies’, a local mushroom grower, was initially collected from local forests. Sawdust of the three types underwent a first pasteurization for 15 min in boiling water at 100 °C to remove any dirt, insects, or any fungal or bacterial contamination. Then, the sawdust was drained and sundried for several days. To prepare each substrate, 4.8 kg of sawdust were mixed with 1.2 kg of wheat bran using a concrete mixer (74,540) to get a homogenous formula. Thereafter, substrate formulas were wetted to a moisture level of 60–70%; when the squeezed substrate expands slowly, no dampness appears on the hand (Green & Mueller, 1995). After wetting, substrate formulas were filled into six autoclavable polypropylene filter patch bags of 1 kg capacity each. The bags were then sealed and placed in modified barrel steamers equipped with a closing pressurized cover, thermometer, pressure gauge, and a faucet, and were subjected to steam pasteurization for 6 h at 120 °C and 10⁴ Pa.

Cooling and spawning

After autoclaving, bags were transferred to a clean and cool room set at 16 °C to reduce the substrate temperature below 25 °C for an appropriate spawning process and suitable mycelium growth (Chang & Miles, 2004). Spawning (at a 2% rate) was carried out in hygienic conditions at the top of substrates using grain spawn of shiitake strain 3782 sourced from Mushies (an online store). Then, the bags were labelled and moved to the incubation room.

Incubation phase

Incubation occurred in complete darkness at a temperature of 20–22 °C and a relative humidity of 50–60%. Temperature and relative humidity inside the incubation room were monitored and kept constant using the humidity/temperature meter (Lutron HT-3007SD). During this phase, five consecutive stages of mycelial development were distinguished, starting with stage 1: mycelia run (thin layer of white hyphae completely covering the block), followed by stage 2: mycelia coat formation (hardening of the mycelia sheet covering the whole substrate surface), stage 3: bump formation (clumps of mycelia developing into popcorn shape), stage 4: browning/pigmentation (mycelia block developing a dark brown and dry outer protective layer), and stage 5: coat hardening (hardening of the dark coat) (Chen, 2001). The end of each stage was recorded in days after spawning (DAS).

Soaking and supplementation

When blocks hardened (stage 5), polyethylene bags were removed, and the substrate blocks were soaked in plastic bags containing ice for 24 h, creating a thermal shock to induce fruiting (Stamets, 1993). With respect to treatments T2, T4, and T6, 5 grams of Lithovit®-Amino 25 were added to 1 L of cold distilled water (concentration 5 g/L) and added to the soaking bag containing ice. The product is a nitrogen-rich fertilizer suitable for use in organic farming (according to Regulation (EC) No. 834/2007-European Community). It is made by tribodynamic activation and micronization of dolomite, and it is composed of 50.0% calcium carbonate (CaCO₃), 28.0% calcium oxide (CaO), 9.0% silicon dioxide (SiO₂), 3.0% total nitrogen (N), 1.8% magnesium oxide (MgO), 0.5% iron (Fe), and 0.02% manganese (Mn). It is obtained by adding (25% admixture) highly energised 16 water-soluble vegetable L-amino acids to Lithovit particles (Bilal, 2010).

Fruit induction and harvest

Twenty-four hours after soaking, the fruiting room was set at a temperature of 16 °C, a relative humidity >90%, and artificial light (2000 lux) to induce fruit formation. High relative humidity was maintained inside the fruiting room using humidifiers (SANI-JET AIR 2836/P0). Two to three days later, pinheads started to appear; thus, blocks were watered daily, three times a day, until mushrooms became fully developed. The date of fruiting was recorded on all treatments as soon as distinct pinheads started to appear on the blocks. Fruit development took around four to five days at the different treatments. Mushrooms were harvested using clean scissors as soon as the gills became visible and the outer edge of the mushroom was slightly curled under (Mudge et al., 2013). Figure 1 below shows the spawn run and fruiting stages.

The date of harvest was then recorded in DAS. After the first harvest, the blocks were put again in dark conditions for around 2 weeks. Two weeks later, blocks were soaked for a second time in ice for all treatments to induce a second harvest. The Fig. 2 below shows the readiness of mushrooms for harvest.

Mushroom number at first (MN1) and second (MN2) harvests and mushroom weight (MW1) and second (MW2) harvests were recorded on the total number of mushrooms harvested per bag. Biological yield corresponded to the total weight of mushrooms harvested per bag at harvest 1 (BY1) and harvest 2 (BY2). The total biological yield (TBY) corresponded to the summation of BY1 and BY2. Biological efficiency (BE) was calculated for each treatment according to Yang, Guo & Wan (2013), as follows:

$$BE\left(\mathrm{\%}\right)={\displaystyle \frac{totalbiologicalyield\left(g/bag\right)}{initialdryweightofsubstrate\left(g\right)x100}}$$

Further, the following physical characteristics were assessed on mushrooms obtained at harvest 1 using a sliding caliper: pileus diameter (PD), pileus thickness (PT), stipe diameter (SD), and stipe length (SL), and the ratio PD/SL was calculated. Mushroom firmness was measured using a setamatic penetrometer (Stanhope-Seta) on five different points of the pileus and the average value was calculated for each sample.

Evaluation of mushroom composition

Various analytical tests were conducted on representative fresh samples of each treatment to evaluate their nutritional composition. A moisture analyzer (M5-Thermo A64M) was used to evaluate the moisture content. For the determination of ash content, five grams of macerated mushroom were heated in a muffle furnace (Carbolite Furnace OAF 10/1) at 550 °C for 24 h, and the ash content was calculated as follows:

$$Ash\left(g\right)=W_2\left(g\right)-W_1\left(g\right)$$

where W2: weight of the crucible containing ash, and W1: weight of an empty crucible.

Fat content was determined using the Soxhlet apparatus technique (Luque de Castro & García Ayuso, 2000) as follows: 10 g of mushrooms were macerated with sea sand and placed in the extractor. The round bottom flask of the extractor was weighed (W₁), then filled with 500 mL of hexane and heated. The cycle was repeated several times for 12 h, during which hexane gradually evaporated and the extracted fat remained at the flask bottom. The flask containing the extracted fat was weighed (W₂). Fat content was calculated and then expressed in percentage:

$$Fat\left(g\right)=W_2\left(g\right)-W_1\left(g\right)$$

Crude protein content (N × 4.38) (%) was determined using the macro-Kjeldhall method based on Reis et al. (2012). Crude fiber content (%) was determined according to Association of Official Chemistry (AOAC) (1982) as the loss of ignition of dried residue remaining after digestion of the mushroom sample with 1.25% (w/v) H₂HSO₄ and 1.25% (w/v) NaOH. Carbohydrates content was then calculated as follows:

$$\begin{array}{rl}\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{s}\left(\mathrm{\%}\right)=& 100-(\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{e}\mathrm{r}\left(\mathrm{\%}\right)+\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\left(\mathrm{\%}\right)\\ & +\mathrm{f}\mathrm{a}\mathrm{t}\left(\mathrm{\%}\right)+\mathrm{m}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\left(\mathrm{\%}\right))\end{array}$$

Vitamin C as ascorbic acid (mg/100 g) was determined titrametrically using 2.6 Dichlorophenolindophenol methods based on Tibuhwa (2014). A 5% metaphosphoric acid solution was added to a known weight of grounded sample, and the mixture was stirred for 30 min. The mixture was then filtered through Whatman No. 42 filter paper using a suction pump. 10 mL of the filtrate were pipetted into a 250 mL conical flask and titrated with 0.025% of 2.6 Dichlorophenolindophenol reagents. The amount of vitamin C in each extract was calculated using the following equation:

$$mgofascorbicacidper100g={\displaystyle \frac{AxIx{V}_{1}x100}{V_{2}xW}}$$

where A = quantity of ascorbic acid (mg) reacting with 1 ml of 2.6 indophenol;

I = volume of indophenol (in mL) required for the completion for the titration with extract;

V₁ = total volume of extract;

V₂ = volume of extract used for each titration.

W = weight of the mushroom sample extracted

Vitamin D₂ as ergocalciferol and vitamin D₃ as cholecalciferol (in µg/100 g) were determined by high performance liquid chromatography (HPLC) according to Mattila et al. (1994). Analytical tests on mushrooms were performed in triplicates.

Statistical analysis

Data analysis applied the two-way ANOVA (factorial ANOVA) and means were compared by Duncan’s Multiple Range test at P_value < 0.05 using SPSS 25 program version 26. Also, an independent sample t-test compared production between harvests 1 and 2. Stepwise regression evaluated the relationship between the total biological yield (dependent variable) and the productive indicators (MN1 and MN2, MW1 and MW2, BY1 and BY2, and PD, PT, SD, and SL) as independent variables.

Sawdust properties

Analysis of substrate properties (Table 1) showed that MS had a higher C/N ratio, thus a lower nitrogen content compared to OS and AS. Crude protein content was the highest in OS, followed by AS and MS (3.04, 2.84, and 1.59%, respectively). Also, MS was the richest in carbohydrates and the poorest in ash content, while OS was the richest in ash and the poorest in carbohydrates. While lignin content was lower in MS and AS than in OS (29.2, 21.6, and 17.7%, respectively), hemicellulose content was higher in both sawdust types than in OS (37.1, 16.4, and 9.1%, respectively). In addition, cellulose content was the highest in AS and lower in MS compared to OS (50.2, 29.2, and 38.8%, respectively). Sawdust pH was higher in MS and AS than in OS (6.6., 6.7, and 5.4, respectively).

Shiitake growth on different substrates

Results (Table 2) showed that in comparison to the control (T1), there was a significant delay in the dates of the different stages of mycelia growth: stages 1, 2, 3, 4, and 5, in T3, T5, and T6, and in the stages 2, 4, and 5 recorded in T4. In particular, the last stage of growth (stage 5) showed respective delays of 6.6, 4.8, 9.3, and 5.6 d in T3, T4, T5, and T6 compared to T1. All stages of mycelia development were almost achieved simultaneously on maple and apple sawdust.

Findings in Fig. 3 showed a significant difference in the dates of fruit formation and harvest in supplemented maple and apple sawdust compared to non-supplemented cases, with an earliness in both dates in supplemented cases; by 4.1 and 3.6 d, respectively, in T4 compared to T3, and 5.4 and 5.2 d, respectively, in T6 compared to T5. Also, there was a slight difference in harvest date recorded on supplemented oak sawdust compared to non-supplemented substrate: earliness of 3.0 d. However, whether the three sawdust were supplemented or not, harvest on maple and apple sawdust was significantly delayed compared to oak sawdust. The delay in harvest ranged between 7.7 and 8.3 d on maple sawdust and between 10.5 and 12.7 d on apple sawdust compared to oak sawdust.

Shiitake production in different treatments

Results in Table 2 showed that the separate effect of the factor ‘substrate’ and the combined effect of both factors ‘substrate x supplementation’ were statistically significant (P_value < 0.05) on MN1 and MN2, MW1 and MW2, and the biological yield obtained at the second harvest (BY2). However, the separate effect of the factor ‘supplementation’ was significant on all productive indicators except MN2.

Compared to T1, the average mushroom number was significantly lower, and the average mushroom weight was significantly higher in all treatments at both harvests.

At the first harvest, MN1 decreased significantly by 18.2 and 4.9 mushrooms on average, and MW1 increased by 30.4 and 15.4 grams in supplemented oak and apple sawdust (T2 and T6), respectively, compared to non-supplemented substrates (T1 and T5). At the same harvest, average BY1 had a significant improvement in all supplemented oak, maple, and apple sawdust compared to non-supplemented ones (by around 21.5, 11.4, and 20.3% in T2, T4, and T6, respectively).

At the second harvest, MN2 increased significantly in supplemented maple and apple sawdust compared to non-supplemented substrates (by 3.7 mushrooms on average in T4 and by 8.0 mushrooms in T6). At the same harvest, MW2 increased by around 16.5 and 2.9 g in supplemented oak and maple sawdust (T2 and T4) compared to non-supplemented substrates (T1 and T3). At this harvest, BY2 showed a significant increase in supplemented maple and apple sawdust compared to non-supplemented cases (by around 36.4 and 27.4% in T4 and T6, respectively).

The highest BE was recorded on the supplemented oak sawdust substrate (T2: 59.7%). Supplementation caused a significant improvement in BE in all supplemented vs. non-supplemented sawdust. The BE obtained in supplemented maple sawdust (T4) was comparable to T1 (53.5%), although that obtained in non-supplemented maple sawdust (T3) was significantly lower than that of T1 (42.2%).

Variation in production in each treatment

Comparing the variation in productive indicators between the first and second harvests (Table 3), it was found that the average MN2 significantly decreased compared to the average MN1 in T1 (by 5.5), T3 (by 3.5), and T5 (by 5.5), while it increased significantly in T6 (by 6.5). MW2 decreased significantly with respect to the treatments T2 (by 11.1 g), T3 (by 7.1 g), and T6 (by 16.0 g). Also, the biological yield showed a significant decline in the second harvest in T3, T4, T5, and T6 (by 55.2, 37.6, 58.4, and 54.3%, respectively). Therefore, production was more consistent among consecutive harvests only on substrates containing oak sawdust, while it decreased progressively throughout consecutive harvests on maple and apple sawdust, whether supplemented or not.

Mushrooms’ physical characteristics

Results in Table 4 showed that the factor ‘substrate’ had a significant effect (P value < 0.05) on pileus diameter and mushroom firmness, while the factor ‘supplementation’ had only a significant effect on pileus diameter. The combined factors significantly affected pileus thickness and the ratio PD/SL. PD was significantly higher in T2 compared to all treatments and increased by 1.4 cm compared to T1. PT increased significantly in T4 compared to T3 and T1 (1.6, 1.0, and 1.2 cm, respectively). The SD was comparable on all tested substrates and ranged from 0.6 to 1.2 cm. The ratio PD/SL was significantly lower in T6 compared to control (1.5 and 1.9, respectively). Mushroom firmness was significantly lower in T5 compared to T2 and T4, but it was comparable to control in all treatments, ranging from 4.2 to 5.5 mm.

Correlation of total biological yield and productive indicators

Results of stepwise regression (Table 5) showed that the TBY in T1, T5, and T6 was the most positively affected by the variation in the biological yield obtained at the first harvest (R² = 0.94, 0.99, and 0.96, respectively). In T2, TBY was positively affected by the PD (R² = 0.82); thus, a higher PD obtained at the first harvest following the application of nano-amino will eventually increase the total production on oak sawdust substrate. In T4, TBY was positively affected by the MW2 (R² = 0.96), meaning that heavier mushrooms obtained at the second harvest will enhance the total production of supplemented maple sawdust.

Shiitake nutritional value

Analysis of the mushrooms’ nutritional composition (Table 6) showed that on non-supplemented substrates, shiitake protein content was the highest on AS, followed by MS and OS (9.2, 8.2, and 2.7% in T1, T3, and T5, respectively). Supplementation significantly increased protein content in mushrooms cultivated on the three supplemented sawdust types (5.6, 9.5, and 10.9% in T2, T4, and T6) compared to non-supplemented ones (2.7, 8.2, and 9.2% in T1, T3, and T5). Mushrooms’ crude fiber content increased significantly on supplemented OS and MS compared to non-supplemented cases. On the contrary, it decreased in supplemented than in non-supplemented AS treatments. The fiber content of mushrooms ranged between 1.5 and 3.8%. Fiber content was ameliorated on MS and AS (supplemented or not) compared to the commercially applied oak sawdust-based substrate. Crude fat did not differ significantly among treatments and ranged between 0.3 and 0.4%. Further, ash contents obtained on supplemented OS and AS were significantly higher than those recorded on non-supplemented substrates (0.9 and 1.4% in T2 and T6 compared to 0.6 and 1.1% in T1 and T5, respectively). On the other hand, there was a slight or significant decrease in mushrooms’ carbohydrate contents obtained from supplemented vs. non-supplemented substrates. Though vitamin C contents did not differ because of substrate supplementation, they were significantly higher in mushrooms cultivated on substrates containing AS (11.0–11.2 mg/100 g) than those containing MS (8.0–8.3 mg/100 g) or OS (4.5–4.8 mg/100 g). Vitamin D content showed similar values (<3 µg/L) in all treatments.