Soybean meal, protein extn. residue

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:

05 April 2018 to 20 April 2018

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Nominal amounts of test material (20, 32, 20, 64 and 200 mg) were each separately added to the surface of 20, 10, 2.0, 2.0 and 2.0 litres of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the 3.2, 10, 32 and 100 mg/L loading rate WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through one sheet of filter paper was conducted for the 32 and 100 mg/L loading rates to remove as much dispersed test material as possible. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test material present, however, a particle count conducted on the 100 mg/L loading rate indicated that dispersed test material remained. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.7 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken from the uninoculated control and each loading rate WAF test group at 0 and 72 hours for this analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low aqueous solubility and complex nature of the test material, for the purposes of the study the test medium was prepared as a WAF of the test material. Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.
- Validation of Mixing Period: A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by filtration through a glass wool plus and one sheet of filter paper to remove as much dispersed test material as present. Observations made on the preparations following filtration showed slightly hazy dispersions were formed. It was considered that further filtration at this point would not have removed any more of the dispersed test material present. It is evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: The master cultures were maintained in the laboratory under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C.

ACCLIMATION
- Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.2 - 8.5

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured (TOC): 0 hours:

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: Closed, the flasks were plugged with polyurethane foam bungs to reduce evaporation.
- Fill volume: 100 mL
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.48 x 10^6 cells per mL. Inoculation of 500 mL of test medium with 1.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Culture medium: NaNO3 25.5 mg/L, MgCl2.6H2O 12.16 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.6 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.186 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00327 mg/L, FeCl3.6H2O 0.160 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA.2H2O 0.30 mg/L.
- The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)
- Constantly shaken at approximately 150 rpm for 72 hours.
- The control group was maintained under identical conditions but not exposed to the test material.

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Samples were taken at 26, 50 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to the presence of dispersed test material.
To determine the potential effect of the test material on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- Water Quality Criteria: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.
- Vortex Depth Measurements: The vortex depth was recorded at the start and end of the mixing period.
- Total Organic Carbon Analysis: Analysis of the WAFs was carried out by TOC analysis.

TEST CONCENTRATIONS
- Range finding study: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 L of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through one sheet of filter paper was performed f or the 100 mg/L loading rate to remove as much dispersed test item as possible. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present, however, visual observations showed the 100 mg/L loading rate to be a very pale beige dispersion. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.9 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test item. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated. After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope. It was not possible to monitor algal growth using a Coulter® Multisizer Particle Counter due to the presence of dispersed test material.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.


DATA EVALUATION
- Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (ln Nn – ln N1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = [(Yc – Yt)/ Yc ] x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


DETERMINATION OF ELx VALUES
- For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ELx values were then determined from the equation for the fitted line.
Where appropriate 95 % confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

30 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

42 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

75 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

7.8 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

30 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence limits: 23 to 39 mg/L loading rate WAF

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RANGE-FINDING TEST
- The results showed no effect on growth rate at 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF.
- Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

DEFINITIVE TEST
- Total Organic Carbon Analysis: Total Organic Carbon (TOC) analysis of the test preparations at 0 Hours showed measured concentration to range from less than the LOQ, considered to be 1.0 mg C/L, to 46 mg C/L. A slight decline in concentration was observed at 72 hours in the range of less than the LOQ to 33 mg C/L. The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

- From the study data, it was clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72-Hour exposure period.

- Exponential growth in the control (for algal test): The cell concentration of the control cultures increased by a factor of 218 after 72 hours. This increase was in line with the OECD guideline that states that the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x10^3 cells/mL
Nominal cell density of control at 72 hours: 1.09 x10^6 cells/mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 20 % and hence satisfied the validation criteria given in the OECD guideline which states that this mean must not exceed 35 %.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 3 % and hence satisfied the validation criterion given in the OECD guideline which states that this must not exceed 7 %.

- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L loading rate WAF, however misshapen cells and cell debris was observed to be present in the test cultures at 100 mg/L loading rate WAF.

- Observations on test material solubility: Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of mixing the 1.0 and 3.2 mg/L loading rate WAFs were observed to have formed clear colourless media columns with test material at the surface and a few particles of test material dispersed throughout. The 10, 32 and 100 mg/L loading rate WAFs were observed to have formed hazy media columns, the intensity of which increased with increasing loading rate, a few particles of test material remained at the media surface. After stirring, and following a 1-Hour standing period, the 1.0 mg/L loading rate WAF observed to have formed a clear colourless media column with test material floating at the media surface and dispersed throughout whilst the 10, 32 and 100 mg/L loading rate WAFs were observed to have formed hazy media columns, the intensity of which increased with increasing loading rate, a few particles of the test material remained at the media surface. Given the presence of the dispersed test material in the 3.2, 10, 32 and 100 mg/L loading rate WAFs they were removed by filtration through a glass wool plug. The 32 and 100 mg/L loading rate WAFs were then passed through one sheet of filter paper to remove as much dispersed test material as possible. At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions. The 32 mg/L loading rate WAF test cultures were observed to be pale green dispersions whilst the 100 mg/L loading rate WAF test cultures were observed to have formed hazy dispersions.

Results with reference substance (positive control):

- Results with reference substance valid: Yes. The results from the positive control with potassium dichromate were within the normal ranges for this reference material.
- A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
- EC50
ErC50 (0 to 72 hour): 1.6 mg/L; 95 % confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95 % confidence limits 0.68 to 0.87 mg/L
- Other:
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Reported statistics and error estimates:

- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
- Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 1.0, 3.2 and 10 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 32 mg/L loading rate WAF.
- Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control, 1.0, 3.2 and 10 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05). However, 17 and 30 % inhibition of yield occurred in the 3.2 and 10 mg/L loading rate WAFs respectively which was considered to be significant. As such the NOEL based on yield was considered to be 1.0 mg/L loading rate WAF. Correspondingly the LOEL based on yield was considered to be 3.2 mg/L loading rate WAF.

Table 1: Results of the Total Organic Carbon Analysis

Samples	Nominal Loading Rate (mg/L)	Concentration of TOC (mg C/L)	Concentration of TOC Corrected for Control (mg C/L)
0 Hours	Control	<LOQ	-
	1.0	<LOQ	<LOQ
	3.2	2.94	2.94
	10	4.98	4.98
	32	14.92	14.92
	100	46.22	46.22
72 Hours	Control	<LOQ	-
	1.0	<LOQ	<LOQ
	3.2	1.98	1.98
	10	3.82	3.82
	32	10.82	10.82
	100	33.11	33.11

Vortex Depth Measurement

The vortex depth was recorded at the start of the mixing period and was observed to have formed a dimple at the media surface.

Water Quality Criteria

The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the growth rate EL50 for the test material was 75 mg/L loading rate WAF and the yield EL50 for the test material was 30 mg/L with 95 % confidence limits 23 – 39 mg/L loading rate WAF.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3, under GLP conditions.

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata.

Due to the low aqueous solubility and the complex nature of the test material, for the purpose of the test the test material was prepared as a Water Accomodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flaks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured concentrations to range from less than the limit of quantification (LOQ), considered to be 1.0 mg C/L, to 46 mg C/L. A slight decrease in concentration was observed at 72 hours in the range of less than the LOQ to 33 mg C/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

It was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Under the conditions of this study, the growth rate EL50 for the test material was 75 mg/L loading rate WAF and the yield EL50 for the test material was 30 mg/L with 95 % confidence limits 23 – 39 mg/L loading rate WAF. The NOEL for growth rate and yield were 10 and 1.0 mg/L, respectively. The LOEL for growth rate and yield were 32 mg/L and 3.2 mg/L respectively.

Description of key information

Under the conditions of this study, the growth rate EL50 for the test material was 75 mg/L loading rate WAF and the yield EL50 for the test material was 30 mg/L with 95 % confidence limits 23 – 39 mg/L loading rate WAF. The NOEL for growth rate and yield were 10 and 1.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

75 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3, 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 study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata.

Due to the low aqueous solubility and the complex nature of the test material, for the purpose of the test the test material was prepared as a Water Accomodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flaks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured concentrations to range from less than the limit of quantification (LOQ), considered to be 1.0 mg C/L, to 46 mg C/L. A slight decrease in concentration was observed at 72 hours in the range of less than the LOQ to 33 mg C/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

It was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Under the conditions of this study, the growth rate EL50 for the test material was 75 mg/L loading rate WAF and the yield EL50 for the test material was 30 mg/L with 95 % confidence limits 23 – 39 mg/L loading rate WAF. The NOEL for growth rate and yield were 10 and 1.0 mg/L, respectively. The LOEL for growth rate and yield were 32 mg/L and 3.2 mg/L respectively.