Condensation products of phenol and salicylaldehyde

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June 2016 to 01 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-7.The guidelines related to the study reports for the registration application of pesticide

Version / remarks:

Ref. No. 12-Nousan-8147 on 24 November 2000 & Ref. No.13-Seisan-3986 on 10 October 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Taking into account that concentration below 2 mg/L could not be measured (the lowest concentration of the calibration curve, 2 mg/L was considered to be the quantification limit of the measurement which corresponds about 1 mg carbon/L, analytical measurement was only performed at the concentrations of 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs to justify that the Test Material was present in the test solution, since more accurate data could not be
provided by TOC.

Vehicle:

no

Details on test solutions:

- Because the test material is a polymer, test solutions were prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. The saturated Test Material solutions (mg/L nominal loading rates WAFs) at the four highest test concentrations were prepared individually by dispersing/dissolving the needed amount of Test Material into the test medium (OECD Medium) two days before the start of the treatment.
- These solutions were shaken for about 24 hours at approximately 30 °C and then were equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solutions. Test solutions were distributed into test vessels prior to introduction of algae.
- As the Test Material was considered to be toxic at low concentrations and the lowest concentration could not be prepared individually with a sufficient precision, therefore it was prepared from a serial dilution of the highest concentration to give the 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.

BREEDING
- Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
- The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The preculture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of four days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.6 - 23.5 °c

pH:

7.54 - 8.79

Nominal and measured concentrations:

Nominal: 2.5, 5.0 and 10.0 mg/L WAF
Measured: Below the limit of quantification, 2.39 and 6.53 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: 100 mL
- Flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- The flasks were covered with air-permeable stoppers.
- Initial cells density: Approximately 10^4 cells/mL
- Control end cells density: 69.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
- Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations:
Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/L, MgCl2 × 6 H2O 12.0 mg/L, CaCl2 × 2 H2O 18.0 mg/L, MgSO4 × 7 H2O 15.0 mg/L and KH2PO4 1.6 mg/L.
Stock solution 2 (iron): FeCl3 × 6 H2O 64.0 μg/L and Na2EDTA × 2H2O 100.0 μg/L.
Stock solution 3 (trace elements): H3BO3 185.0 μg/L, MnCl2 × 4 H2O 415.0 μg/L, ZnCl2 3.0 μg/L, CoCl2 × 6 H2O 1.5 μg/L, CuCl2 × 2 H2O 0.01 μg/L and Na2MoO4 × 2 H2O 7.0 μg/L.
Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/L
- Intervals of water quality measurement: Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The pH was checked at the beginning and at the end of the test, in the control and each concentration.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 7905 lux (equivalent to ~107 μE/m^2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test
vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED:
- The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Preliminary Range Finding Tests: Algal cells were exposed to each concentration of the Test Material plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group. Nominal concentrations: 0.1, 1, 10 and 100 mg/L WAF. At all applied test concentrations (including the control) the algal cells were normal, no morphological deviations were observed.
- Test material concentrations in the Definitive Test: Because toxic response was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment. The concentrations in the definitive test were: 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) and 1.25., 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs. Biological results are based on the nominal test material concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

5.07 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 4.52 – 5.68 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.42 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 2.13 – 2.76 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 2.68 – 3.36 mg/L

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

2.5 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1.25 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1.25 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.625 other: % saturated solution (of 10.0 mg/L nominal loading rate WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

and yield

Details on results:

CELL NUMBERS
- The cell number in each flask was determined at the 24th, 48th, 72nd hours. The results of determinations are listed in Table 1.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
- There were no morphological deviations of the algal cell during the study in any of the test groups.

AVERAGE SPECIFIC GROWTH RATES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentration range of 1.25 – 10 mg/L nominal loading rate WAF. At the Test Material concentration 1.25 mg/L nominal loading rate WAF 8.6 % inhibition was observed attaining statistically significance rather to biological variability of the test system then a toxic effect, therefore the LOELR was considered to be 2.5 mg/L nominal loading rate WAF, correspondingly the No Observed Effect Loading Rate (NOELR) was determined as 1.25 mg/L nominal loading rate WAF.
- The 72 h ErC50/ ErL50 value was determined [by Probit analysis (TOXSTAT software)] as 5.07 mg/L nominal loading rate WAF (95 % confidence limits: 4.52 – 5.68 mg/L nominal loading rate WAF).
- Results can be seen in Table 2.

AREAS UNDER THE GROWTH CURVES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value in the concentration range of concentration range of 1.25 – 10 mg/L nominal loading rate WAF, accordingly the No Observed Effect Loading Rate (NOELR) determined as 0.625 % of 10.0 mg/L nominal loading rate WAF.
- The 72 h EbL50 value was determined [by Probit analysis (TOXSTAT software)] as 3.00 mg/L nominal loading rate WAF (95 % confidence limits: 2.68 – 3.36 mg/L mg/L nominal loading rate WAF).
- Results can be seen in Table 3.

YIELD
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value in the tested concentration range of 1.25 – 10 mg/L nominal loading rate WAF, accordingly the No Observed Effect Loading Rate (NOELR) determined as 0.625 % of 10.0 mg/L nominal loading rate WAF.
- The 72 h EyL50 value was determined [by Probit analysis (TOXSTAT software)] as 2.42 mg/L (95 % confidence limits: 2.13 – 2.76 mg/L mg/L nominal loading rate WAF).
- Results can be seen in Table 4.

- The lowest test concentration 0.625 % saturated solution (of 10.0 mg/l nominal loading rate WAF) was excluded from the probit analyses in case of average specific growth rates, areas under the growth curves and yield due to dilution from the highest concentration which resulted in different unit.

Results with reference substance (positive control):

- The date of the last study with the reference material Potassium dichromate was: 27 - 30 October 2015.
- The 72h ErC 50: 0.84 mg/L, (95 % confidence limits: 0.77 – 0.92 mg/L)
- The 72h EbC 50: 0.59 mg/L, (95 % confidence limits: 0.54 – 0.65 mg/L)
- The 72h EyC 50: 0.55 mg/L, (95 % confidence limits: 0.50 – 0.60 mg/L)
- These values are within the range of laboratory ring test data.

Reported statistics and error estimates:

- The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
- The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software.
- The ErC50/ ErL50, EbC50/ EbL50 and EyC50/ EyL50 values of the Test Material and their confidence limits were calculated using Probit analysis by TOXSTAT software. The lowest test concentration 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) was excluded from the Probit analysis due to dilution from the highest concentration which resulted in different unit.
- Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
- For the determination of the LOELR and NOELR, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Validity

- The cell density in the control cultures were increased by the factor of 69.5 within three days.

- The mean coefficient of variation for section-by-section specific growth rates (days 0- 1; 1-2; 2-3) in the control cultures was 7.59 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.93 %.

- All validity criteria were met, therefore the study can be considered as valid.

Table 1: MeanCell Number (× 10^4 cell/mL) determined in the Main Experiment

Concentration	Number of Cells
	0 h	24 h	48 h	72 h
Control	1.00	4.00	17.17	69.50
0.625 % of 10.0 mg/L nominal loading rate WAF	1.00	4.00	17.33	67.67
1.25 mg/L nominal loading rate WAF	1.00	3.67	16.67	48.33
2.5 mg/L nominal loading rate WAF	1.00	3.67	15.53	41.67
5 mg/L nominal loading rate WAF	1.00	3.00	7.33	18.67
10 mg/L nominal loading rate WAF	1.00	1.00	1.00	1.33

 

Table 2: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Concentrations	Growth rate (μ) and % inhibition of μ
	0-24 h		0-48 h		0-72 h
	µ	%	µ	%	µ	%
Control	0.0573	0.0	0.0592	0.0	0.0589	0.0
0.625 % of 10.0 mg/L nominal loading rate WAF	0.0569	0.8	0.0594	-0.4	0.0585	0.6
1.25 mg/L nominal loading rate WAF	0.0538	6.2	0.0586	1.0	0.0539*	8.6
2.5 mg/L nominal loading rate WAF	0.0538	6.2	0.0569	3.9	0.0518*	12.1
5 mg/L nominal loading rate WAF	0.0458	20.1	0.0415*	29.9	0.0405*	31.2
10 mg/L nominal loading rate WAF	0.0000*	100.0	0.0000*	100.0	0.0032*	94.6

*: Statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05).

 

Table 3: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentrations	Area under the Growth Curves (A) and Percentage Inhibition of A
	0-24 h		0-48 h		0-72 h
	A	%	A	%	A	%
Control	36.0	0.0	266.0	0.0	1282.0	0.0
0.625 % of 10.0 mg/L nominal loading rate WAF	36.0	0.0	268.0	-0.8	1264.0	1.4
1.25 mg/L nominal loading rate WAF	32.0	11.1	252.0	5.3	1008.0*	21.4
2.5 mg/L nominal loading rate WAF	32.0	11.1	236.0	11.3	896.0*	30.1
5 mg/L nominal loading rate WAF	24.0	33.3	124.0*	53.4	412.0*	67.9
10 mg/L nominal loading rate WAF	0.0*	100.0	0.0*	100.0	4.0*	99.7

*: Statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05).

Table 4: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentrations	Yield (Y) and % inhibition of Y (0–72 h)
	Y	%
Control	68.5	0.0
0.625 % of 10.0 mg/L nominal loading rate WAF	66.7	2.7
1.25 mg/L nominal loading rate WAF	47.3*	30.9
2.5 mg/L nominal loading rate WAF	40.7*	40.6
5 mg/L nominal loading rate WAF	17.7*	74.2
10 mg/L nominal loading rate WAF	0.3*	99.5

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 0-72 h average specific growth rates was significantly different from that of the control group in the examined concentration range of 2.5 – 10 mg/L nominal loading rates WAFs, therefore the LOELR was determined as 2.5 mg/L nominal loading rate WAF and the NOELR was determined as 1.25 mg/L nominal loading rate WAF. Areas and yield were statistically significantly different from that of the control group in the examined concentration range of 1.25 – 10 mg/L nominal loading rates WAFs, therefore the overall LOELR was determined as 1.25 mg/L nominal loading rate WAF and the NOELR was determined as 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF). The EL50 values were 5.07, 2.42 and 3.00 mg/L nominal loading rate WAF for growth rate, yield and biomass, respectively.

Executive summary:

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The test was performed in accordance with the standardised guidelines OECD 201, EU Method C.3 and OCSPP850.5400, under GLP conditions.

A toxic response was observed during the preliminary range-finding tests, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment. The concentrations in the definitive test were: 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) and 1.25., 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs.

Since analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the Test Material was measured this way (and it was not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning of the experiment to justify that the Test Material was present in the test solution, since more accurate data could not be provided by TOC.

Furthermore taking into account that concentration below 2 mg/L could not be measured (the lowest concentration of the calibration curve, 2 mg/L was considered to be the quantification limit of the measurement which corresponds about 1 mg carbon/L), analytical measurement was only performed at the concentrations of 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs. Therefore biological results are based on the nominal test material concentrations.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The ErL50, EbL50 and EyL50 values of the Test Material and their confidence limits were calculated using Probit analysis by TOXSTAT software. The lowest test concentration 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) was excluded from the Probit analysis due to dilution from the highest concentration which resulted in different unit.

Under the conditions of this study, the 0-72 h average specific growth rates was significantly different from that of the control group in the examined concentration range of 2.5 – 10 mg/L nominal loading rates WAFs, therefore the LOELR was determined as 2.5 mg/L nominal loading rate WAF and the NOELR was determined as 1.25 mg/L nominal loading rate WAF.

Areas and yield were statistically significantly different from that of the control group in the examined concentration range of 1.25 – 10 mg/L nominal loading rates WAFs, therefore the overall LOELR was determined as 1.25 mg/L nominal loading rate WAF and the NOELR was determined as 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF). The EL50 values were 5.07, 2.42 and 3.00 mg/L nominal loading rate WAF for growth rate, yield and biomass, respectively.

Description of key information

Under the conditions of this study, the 0-72 h average specific growth rates was significantly different from that of the control group in the examined concentration range of 2.5 – 10 mg/L nominal loading rates WAFs, therefore the LOELR was determined as 2.5 mg/L nominal loading rate WAF and the NOELR was determined as 1.25 mg/L nominal loading rate WAF. Areas and yield were statistically significantly different from that of the control group in the examined concentration range of 1.25 – 10 mg/L nominal loading rates WAFs, therefore the overall LOELR was determined as 1.25 mg/L nominal loading rate WAF and the NOELR was determined as 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF). The EL50 values were 5.07, 2.42 and 3.00 mg/L nominal loading rate WAF for growth rate, yield and biomass, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.07 mg/L

EC10 or NOEC for freshwater algae:

1.25 mg/L

Additional information

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The test was performed in accordance with the standardised guidelines OECD 201, EU Method C.3 and OCSPP850.5400, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A toxic response was observed during the preliminary range-finding tests, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment. The concentrations in the definitive test were: 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) and 1.25., 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs.

Since analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the Test Material was measured this way (and it was not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning of the experiment to justify that the Test Material was present in the test solution, since more accurate data could not be provided by TOC.

Furthermore taking into account that concentration below 2 mg/L could not be measured (the lowest concentration of the calibration curve, 2 mg/L was considered to be the quantification limit of the measurement which corresponds about 1 mg carbon/L), analytical measurement was only performed at the concentrations of 2.5, 5.0 and 10.0 mg/L nominal loading rates WAFs. Therefore biological results are based on the nominal test material concentrations.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErL50, EbL50 and EyL50 values of the Test Material and their confidence limits were calculated using Probit analysis by TOXSTAT software. The lowest test concentration 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF) was excluded from the Probit analysis due to dilution from the highest concentration which resulted in different unit.

Under the conditions of this study, the 0-72 h average specific growth rates was significantly different from that of the control group in the examined concentration range of 2.5 – 10 mg/L nominal loading rates WAFs, therefore the LOELR was determined as 2.5 mg/L nominal loading rate WAF and the NOELR was determined as 1.25 mg/L nominal loading rate WAF.

Areas and yield were statistically significantly different from that of the control group in the examined concentration range of 1.25 – 10 mg/L nominal loading rates WAFs, therefore the overall LOELR was determined as 1.25 mg/L nominal loading rate WAF and the NOELR was determined as 0.625 % saturated solution (of 10.0 mg/L nominal loading rate WAF). The EL50 values were 5.07, 2.42 and 3.00 mg/L nominal loading rate WAF for growth rate, yield and biomass, respectively.