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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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:
Experimental Starting Date: 24 May 2012, Experimental Completion Date: 22 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Definitive test: 62.5, 125, 250, 500 and 1000 mg active ingredient (ai)/L

- Sampling method:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours.

- Sample storage conditions before analysis:
The samples were stored at approximately -20 °C prior to analysis.
Vehicle:
no
Details on test solutions:
RANGE-FINDING TEST:
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg ai/L for a period of 96 hours. All test concentrations were corrected for a test item water content of 8.7%.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (548 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 500 mL to give a 1000 mg ai/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 100, 10 and 1.0 mg ai/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (23 mL) to give the required test concentrations of 1.0, 10, 100 and 1000 mg ai/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.

After 96 hours the cell density of each flask was determined using a Coulter Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

DEFINITIVE TEST:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 62.5, 125, 250, 500 and 1000 mg/L.

Experimental Preparation:
For the purpose of the definitive test, the test item was dissolved directly in culture medium. All test concentrations were corrected for a test item water content of 8.7%.

An amount of test item (2192 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 2 liters to give a 1000 mg ai/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 500, 250, 125 and 62.5 mg ai/L. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (11.4 mL) to give the required test concentrations of 62.5, 125, 250, 500 and 1000 mg ai/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours
















Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

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 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10E4 – 10E5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable.
Test temperature:
Temperature was maintained at 24 ± 1°C throughout the test (the temperature within the incubator was recorded daily).
pH:
The culture medium pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
The pH of the control and each test preparation was determined at initiation of the test and after 96 hours exposure. The pH was measured using a WTW pH 320 pH meter.
Nominal and measured concentrations:
Definitive test:
Nominal concentrations: 62.5, 125, 250 and 1000 mg/L.
Measured concentrations: near nominal concentrations (see results section).
Details on test conditions:
DEFINITIVE TEST EXPOSURE CONDITIONS:
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 8.75 x 10E5 cells per mL. Inoculation of 1 liter of test medium with 11.4 mL of this algal suspension gave an initial nominal cell density of 1 x 10E4 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.

GROWTH MEDIUM
CUlture medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined below:
NaNO3: 25.5 mg/l
MgCl2.6H2O: 12.164 mg/l
CaCl2.2H2O: 4.41 mg/l
MgSO4.7H2O: 14.7 mg/l
K2HPO4 1.044: mg/l
NaHCO3 15.0: mg/l
H3BO3 0.1855: mg/l
MnCl2.4H2O: 0.415 mg/l
ZnCl2: 0.00327 mg/l
FeCl3.6H2O: 0.159 mg/l
CoCl2.6H2O: 0.00143 mg/l
Na2MoO4.2H2O: 0.00726 mg/l
CuCl2.2H2O: 0.000012 mg/l
Na2EDTA.2H2O: 0.30 mg/l
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

RE-GROWTH TEST:
A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item. Aliquots were removed from each replicate control and test culture (0.25 mL and 0.50 mL respectively) and the replicates pooled. Fresh sterile culture medium (100 mL) was added to each to ensure that the test concentration was reduced to below the inhibiting level.

The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron Version 2 Incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 48 hours.




Reference substance (positive control):
yes
Remarks:
A positive control used zinc chloride as the reference item at concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
780 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1 400 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
290 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL 260 - 330 mg ai/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
460 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL 400 - 520 mg ai/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
270 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 24 - 320 mg ai/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
360 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 320 - 420 mg ai/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
Observations on cultures:
All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility:
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control, 62.5, 125, 250 and 500 mg ai/L test cultures were observed to be green dispersions whilst the 1000 mg ai/L test cultures were observed to be pale green dispersions.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 (see attached background material).

The results showed no effect on growth at the test concentrations of 1.0, 10 and 100 mg ai/L. However, growth was observed to be reduced at 1000 mg ai/L.

Based on this information test concentrations of 62.5, 125, 250, 500 and 1000 mg ai/L were selected for the definitive test.

Chemical analysis of the 100 and 1000 mg ai/L test samples at 0 and 96 hours (see Appendix 4 - attached background material) showed near nominal test concentrations were obtained indicating that the test item was stable over the test duration.

Definitive Test

Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 5.

See attached background material for Tables 1 -5.

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. See attached background material for Figures 1 -7.

Growth data

From the data given in Tables 2, 4 and 5, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 96-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate:

ErC10 (0 - 72 h) : 210 mg ai/L

ErC20 (0 - 72 h) : 340 mg ai/L

ErC50 (0 - 72 h) : 780 mg ai/L

ErC10 (0 - 96 h) : 390 mg ai/L

ErC20 (0 - 96 h) : 630 mg ai/L

ErC50 (0 - 96 h) : 1400 mg ai/L*

*Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate.

It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations 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). After 72 hours there were no statistically significant differences between the control, 62.5 and 125 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 125 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 250 mg ai/L. After 96 hours there were no statistically significant differences between the control, 62.5, 125 and 250 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 250 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 500 mg ai/L.

Inhibition of yield:

EyC10 (0 - 72 h) : 140 mg ai/L

EyC20 (0 - 72 h) : 180 mg ai/L

EyC50 (0 - 72 h) : 290 mg ai/L; 95% confidence limits 260 - 330 mg ai/L

EyC10 (0 - 96 h) : 180 mg ai/L

EyC20 (0 - 96 h) : 250 mg ai/L

EyC50 (0 - 96 h) : 460 mg ai/L; 95% confidence limits 400 - 520 mg ai/L

Statistical analysis of the yield data at 72 and 96 hours was carried out as for growth rate. After 72 and 96 hours there were no statistically significant differences between the control, 62.5 and 125 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 125 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 250 mg ai/L.

Inhibition of biomass integral:

EbC10 (0 - 72 h) : 130 mg ai/L

EbC20 (0 - 72 h) : 170 mg ai/L

EbC50 (0 - 72 h) : 270 mg ai/L; 95% confidence limits 240 - 320 mg ai/L

EbC10 (0 - 96 h) : 140 mg ai/L

EbC20 (0 - 96 h) : 200 mg ai/L

EbC50 (0 - 96 h) : 360 mg ai/L; 95% confidence limits 320 - 420 mg ai/L

Statistical analysis of the biomass integral data at 72 and 96 hours was carried out for growth rate. After 72 and 96 hours there were no statistically significant differences between the control, 62.5 and 125 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 125 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 250 mg ai/L.

Physico-chemical measurements:

The pH values of the control and each test preparation are given in Table 2 (attached background material). Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.8 at 0 hours to pH 8.0 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH units after 96 hours and therefore was within the limits given in the Test Guidelines.

Re-growth test:

Re-growth was observed to have occurred in the control and all test cultures after 48 hours. These results indicate that the test item was algistatic in effect.

Verification of test concentrations:

Chemical analysis of the test preparations at 0 and 96 hours (see Appendix 4 - attached background material) showed measured test concentrations to be near nominal with the exception of the 62.5 and 125 mg ai/L test preparations at 0 hours where measured concentrations of 66% and 67% of nominal respectively were obtained. Analysis of duplicate samples which had been stored at approximately -20 °C showed measured concentrations of 91% and 67% of nominal for the 62.5 and 125 mg ai/L test preparations respectively. Given the variability seen at 62.5 mg ai/L and the failure to obtain near nominal concentrations at 125 mg ai/L, samples taken at 24, 48 and 72 hours were also analyzed. Near nominal concentrations were obtained from the 62.5 mg ai/L test preparations at 24, 48 and 72 hours indicating that the initial low measured concentration was anomalous. Analysis of the 125 mg ai/L test preparations at 24, 48 and 72 hours showed measured test concentrations of 102%, 51% and 100% of nominal respectively were obtained. Inspection of the analytical data could show no cause for the variability observed. Given that near nominal concentrations were observed on three of the five sampling occasions (24, 72 and 96 hours) it was considered that the test system had been correctly dosed and that the failure to obtain near nominal concentrations on two occasions was due an unknown analytical issue. As such it was considered appropriate to calculate the result based on the nominal test concentrations only.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and gave the following results:

Growth Rate:
72 h EC50: 780 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 1400 mg ai/L
96 h NOEC: 250 mg ai/L

Yield:
72 h EC50: 290 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 460 mg ai/L
96 h NOEC: 125 mg ai/L

Biomass:
72 h EC50: 270 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 360 mg ai/L
96 h NOEC: 125 mg ai/L


Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 62.5, 125, 250, 500 and 1000 mg active ingredient (ai)/L (three replicate flasks per concentration) for 96 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 Coulter Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Time Point (Hours)

Response Variable

EC50(mg ai/L)

95% Confidence Limits (mg ai/L)

No Observed Effect Concentration (NOEC) (mg ai/L)

Lowest Observed Effect Concentration (LOEC) (mg ai/L)

72

Growth rate

780

*

125

250

Yield

290

260 – 330

125

250

Biomass

270

240 – 320

125

250

96

Growth rate

1400**

*

250

500

Yield

460

400 – 520

125

250

Biomass

360

320 – 420

125

250

 

* It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits.  

** Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate. 

Chemical analysis of the test preparations at 0 and 96 hours showed measured test concentrations to be near nominal with the exception of the 62.5 and 125 mg ai/L test preparations at 0 hours where measured concentrations of 66% and 67% of nominal respectively were obtained. Analysis of duplicate samples which had been stored at approximately -20 °C showed measured concentrations of 91% and 67% of nominal for the 62.5 and 125 mg ai/L test preparations respectively. Given the variability seen at 62.5 mg ai/L and the failure to obtain near nominal concentrations at 125 mg ai/L, samples taken at 24, 48 and 72 hours were also analyzed. Near nominal concentrations were obtained from the 62.5 mg ai/L test preparations at 24, 48 and 72 hours indicating that the initial low measured concentration was anomalous. Analysis of the 125 mg ai/L test preparations at 24, 48 and 72 hours showed measured test concentrations of 102%, 51% and 100% of nominal respectively were obtained. Inspection of the analytical data could show no cause for the variability observed. Given that near nominal concentrations were observed on three of the five sampling occasions (24, 72 and 96 hours) it was considered that the test system had been correctly dosed and that the failure to obtain near nominal concentrations on two occasions was due an unknown analytical issue. As such it was considered appropriate to calculate the result based on the nominal test concentrations only.

A re-growth test was performed which showed the test item to be algistatic in effect.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and gave the following results:
Growth Rate:
72 h EC50: 780 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 1400 mg ai/L
96 h NOEC: 250 mg ai/L
Yield:
72 h EC50: 290 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 460 mg ai/L
96 h NOEC: 125 mg ai/L
Biomass:
72 h EC50: 270 mg ai/L
72 h NOEC: 125 mg ai/L
96 h EC50: 360 mg ai/L
96 h NOEC: 125 mg ai/L

Key value for chemical safety assessment

EC50 for freshwater algae:
780 mg/L
EC10 or NOEC for freshwater algae:
125 mg/L

Additional information

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 62.5, 125, 250, 500 and 1000 mg active ingredient (ai)/L (three replicate flasks per concentration) for 96 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 Coulter Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Time Point (Hours)

Response Variable

EC50(mg ai/L)

95% Confidence Limits (mg ai/L)

No Observed Effect Concentration (NOEC) (mg ai/L)

Lowest Observed Effect Concentration (LOEC) (mg ai/L)

72

Growth rate

780

*

125

250

Yield

290

260 – 330

125

250

Biomass

270

240 – 320

125

250

96

Growth rate

1400**

*

250

500

Yield

460

400 – 520

125

250

Biomass

360

320 – 420

125

250

 

* It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits.  

** Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate. 

Chemical analysis of the test preparations at 0 and 96 hours showed measured test concentrations to be near nominal with the exception of the 62.5 and 125 mg ai/L test preparations at 0 hours where measured concentrations of 66% and 67% of nominal respectively were obtained. Analysis of duplicate samples which had been stored at approximately -20 °C showed measured concentrations of 91% and 67% of nominal for the 62.5 and 125 mg ai/L test preparations respectively. Given the variability seen at 62.5 mg ai/L and the failure to obtain near nominal concentrations at 125 mg ai/L, samples taken at 24, 48 and 72 hours were also analyzed. Near nominal concentrations were obtained from the 62.5 mg ai/L test preparations at 24, 48 and 72 hours indicating that the initial low measured concentration was anomalous. Analysis of the 125 mg ai/L test preparations at 24, 48 and 72 hours showed measured test concentrations of 102%, 51% and 100% of nominal respectively were obtained. Inspection of the analytical data could show no cause for the variability observed. Given that near nominal concentrations were observed on three of the five sampling occasions (24, 72 and 96 hours) it was considered that the test system had been correctly dosed and that the failure to obtain near nominal concentrations on two occasions was due an unknown analytical issue. As such it was considered appropriate to calculate the result based on the nominal test concentrations only.

A re-growth test was performed which showed the test item to be algistatic in effect.