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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2018 to 11 March 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:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2017
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: Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law)
Version / remarks:
1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).
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:
- Analytical measurement was performed at the control and at the applied test concentration levels and from the control at the beginning of the test and in 24 hour intervals thereafter during the experiment.
- Determination of test substance concentrations was performed at each test concentration level and from the additional samples without algae in order to distinguish degradation and adsorption of the test item to algae.
Vehicle:
no
Details on test solutions:
- Because the test material is poorly soluble in water, a test material stock solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
- A saturated test material solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (OECD Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100.0 % saturated solution.
- The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.
- Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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.
- Method of cultivation: 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 pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three 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:
22.4 – 22.9 °C
pH:
7.15 – 8.31
Nominal and measured concentrations:
- Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.
- Measured geometric mean concentrations: 2.08, 3.28, 5.75, 12.39 and 62.78 mg/L in presence of algae and 4.67, 9.11, 16.84, 41.22 and 85.17 mg/L in absence of algae
- Initial measured concentrations: 3.38, 7.95, 15.4, 39.2 and 80.7 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks covered with air-permeable stoppers.
- Material, size, headspace, fill volume: 100 mL
- Agitation: Continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Initial cells density: approximately 10^4 algal cells per mL test medium.
- No. of vessels per concentration: 3
- No. of vessels per vehicle control: 6
- There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to
distinguish degradation and adsorption of the test material to algae.

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, MgCl2.6H2O 12.0 mg/L, CaCl2.2H2O 18.0 mg/L, MgSO4.7H2O 15.0 mg/L and KH2PO4 1.6 mg/L.
- Stock solution 2 (iron): FeCl3.6H2O 64.0 µg/L and Na2EDTA.2H2O 100.0 µg/L.
- Stock solution 3 (trace elements): H3BO3 185.0 µg/L, MnCl2.4H2O 415.0 µg/L, ZnCl2 3.0 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L and Na2MoO4.2H2O 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
- 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 7813 lux (equivalent to 106 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:
- Determination of cell concentrations: Counting chamber
- 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 24, 48 and 72 h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Separation factor of 2.0
- Range finding study test concentrations: 0.1, 1, 10 and 100 % saturated solutions
A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. 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.
- Results used to determine the conditions for the definitive study: Yes. Because significant inhibition was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main test.

CALCULATIONS
- Calculation of Average Specific Growth Rate:
Concentration-effect relationship was calculated by comparing growth rates in control, test cultures in the following way. The average specific growth rate (μ) for individual cultures are calculated from the following relationship:

µ = [ln(Nn) - ln(N0)] / tn – t0

Where
ln (Nn) = natural logarithm of measured number of cells/mL at time tn
ln (N0) = natural logarithm of measured number of cells/mL at time t0
t0 = time (hour) of the beginning of the test
tn = time (hour) of nth measurements after the beginning of the test

The percentage inhibition of growth rate = % Iµ:

% Iµ= [(µc - µt) / µc ] ·100 %

Where
% Iμ = percent inhibition in average specific growth rate
μc = mean growth rate of the control
μt = mean growth rate of test concentration t

- Calculation of Area Under the Growth Curve:

A = [(N1 – N0) / 2] · t1 + [(N1 + N2 – 2N0) / 2] · (t2 – 21) + [(Nn-1 + Nn – 2N0) / 2] · (tn – tn – 1)

Where
N0 = nominal number of cells/mL at time t0 (start of the test)
N1 = mean measured number of cells/mL at t1 (24 hours)
N2 = mean measured number of cells/mL at t2 (48 hours)
Nn = mean measured number of cells/mL at tn
t1 = time of first measurement after start of the test
t2 = time of second measurement after start of the test
tn = time of nth measurement after start of the test

The percentage inhibition of area = % IA

% IA = [(Ac – At) / Ac] · 100 %

Where % IA = percent inhibition in area under the growth curve
Ac = mean area of the control
At = mean area of test concentration t

- Calculation of Yield:
Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated.

Percentage inhibition in yield = % Iy

% Iy = [(yc – yi) / yc] · 100

Where:
yc = mean value for yield in the control group
yi = mean value for yield for the test concentration
Area under the growth curve (biomass), average specific growth rate and yield were calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
48.16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits 39.50 – 58.72
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.95 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
biomass and yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
15.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
biomass and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.37 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. limits 14.61 – 18.35
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18.99 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits 16.83 – 21.44
Details on results:
CONCENTRATIONS OF THE TEST MATERIAL
- The test material could only be measured at the highest concentration level of 100.0 % saturated solution (nominal) 72 hours after the treatment (at the end of the experiment) in presence of algae.
- In order to calculate the mean exposure concentrations, where the measured concentration was below the Quantification Limit, the concentration was taken as half of the Limit of Quantification (LOQ = 1.0 mg/L). Therefore the corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae, while the measured geometric mean test material concentrations were: 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L in absence of algae.
- According to the relevant guideline (OECD 201) disappearance of the test material from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test material in the course of the test was accompanied by a decrease in growth inhibition. If not, it may be appropriate to base the analysis of the results on initial (nominal or measured) concentrations.
- Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system. The specific growth rate for all of the tested concentrations was not clearly reduced at the section days 2-3 and it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.
- Therefore it is recommended to express the EC50 and NOEC/LOEC values in nominal concentrations and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

CELL NUMBERS
- The cell number in each flask (three replicates per treated group) was determined at the 24th, 48th, 72nd hours.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
- Chuff cells were observed at the highest concentration level of 100.0 % saturated solution 48 and 72 hours after the treatment.

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 initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 48.16 mg/L (95 % confidence limits: 39.50 – 58.72 mg/L (measured, initial).

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 initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 18.99 mg/L (95 % confidence limits: 16.83 – 21.44 mg/L (measured, initial).

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 initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 16.37 mg/L (95 % confidence limits: 14.61 – 18.35 mg/L (measured, initial).
Results with reference substance (positive control):
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate
satisfactory test conditions.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 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 (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the calculated geometric mean concentrations).
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 LOEC and NOEC, 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 increased by a factor of 67.50 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 7.17 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.72 %.

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

Table 1: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Concentration

Growth rate (μ) and % inhibition of μ

Nominal [% sat. sol.]

Measured [mg/L]

0-24 h

0-48 h

0-72 h

µ

%

µ

%

µ

%

Control

0.00

0.0573

0.0

0.0586

0.0

0.0585

0.0

6.25

3.38

0.0569

0.8

0.0590

-0.7

0.0586

-0.2

12.5

7.95

0.0538

6.2

0.0590

-0.7

0.0585

-0.1

25.0

15.4

0.0529

7.8

0.0384*

34.5

0.0373*

36.3

50.0

39.2

0.0498

13.2

0.0384*

40.6

0.0328*

44.0

100.0

80.7

0.0345*

39.8

0.0173*

70.5

0.0240*

58.9

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

 

Table 2: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration

Area under the Growth Curves (A) and Percentage Inhibition of A

Nominal [% sat. sol.]

Measured [mg/L]

0-24 h

0-48 h

0-72 h

A

%

A

%

A

%

Control

0.00

36.0

0.0

260.0

0.0

1246.0

0.0

6.25

3.38

36.0

0.0

264.0

-1.5

1260.0

-1.1

12.5

7.95

32.0

11.1

256.0

1.5

1248.0

-0.2

25.0

15.4

32.0

11.1

128.0*

50.8

356.0*

71.4

50.0

39.2

28.0

22.2

108.0*

58.5

276.0*

77.8

100.0

80.7

16.0*

55.6

48.0*

81.5

120.0*

90.4

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

 

Table 3: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration

Yield

Nominal [% sat. sol.]

Measured [mg/L]

0-72 h

Y

%

Control

0.00

66.5

0.0

6.25

3.38

67.0

-0.8

12.5

7.95

66.7

-0.3

25.0

15.4

13.7*

79.4

50.0

39.2

9.7*

85.5

100.0

80.7

4.7*

93.0

*: 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, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.
Executive summary:

The potential of the test material to cause aquatic toxicity to algae was examined in accordance with the standardised guidelines OECD 201, EU Method C.3., EPA OCSPP 850.5400 and JMAFF guidelines, under GLP conditions.

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.

A significant toxic response was observed during the preliminary range-finding test, 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 nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated control. There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test material to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae. In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test material concentrations of 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution, respectively.

Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.

Therefore it is recommended to express the EC50 and NOEC/LOEC values in the initial test material concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

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 ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) 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, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
48.16 mg/L
EC10 or NOEC for freshwater algae:
7.95 mg/L

Additional information

The potential of the test material to cause aquatic toxicity to algae was examined in accordance with the standardised guidelines OECD 201, EU Method C.3., EPA OCSPP 850.5400 and JMAFF guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

A significant toxic response was observed during the preliminary range-finding test, 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 nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated control. There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test material to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae.

In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test material concentrations of 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution, respectively.

Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.

Therefore it is recommended to express the EC50 and NOEC/LOEC values in the initial test material concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

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 ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.