Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

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:

26 February 2018 to 01 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: JMAFF 2-7-7

Version / remarks:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Version / remarks:

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) 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

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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- A stock solution with a nominal concentration of 40.0 mg/L was prepared with the test material and algal growth medium (OECD Medium) using ultrasonic bath (30 - 40 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

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:

22.5 – 22.9 °C

pH:

7.26 – 8.80

Nominal and measured concentrations:

Nominal: 0.1024, 0.256, 0.64, 1.6 and 4.0 mg/L
Measured geometric mean concentrations: 0.03; 0.05; 0.08; 0.49 and 3.31 mg/L in the presence of algae and 0.063; 0.224; 0.53; 1.51 and 3.98 mg/L in absence of algae.

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 control: 6

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 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Separation factor of 2.5
- Range finding study test concentrations: 0.1, 1, 10 and 100 mg/L
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 inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.5) 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.:

0.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 0.58 – 0.70 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 0.39 – 0.48 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 0.39 – 0.46 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.224 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield and biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield and biomass

Details on results:

CONCENTRATIONS OF THE TEST MATERIAL
- Measured concentrations deviated more than 20 per cent from the nominal at the three lower concentration levels of 0.1024, 0.256, 0.64 mg/L (nominal) at the 24 hours measurements, while the test material could not be detected later during the experiment (48 and 72 hours) in presence of algae.
- The test material was measured close to the nominal in samples containing algae at the start of experiment and after 24 and 48 hours at the two highest concentration levels of 1.6 and 4.0 mg/L (nominal). After 72 hours (at the end of the experiment) the test material could only be measured at the highest concentration level of 4.0 mg/L (nominal), while the measured concentration was also below the Limit of Detection (LOD) at the concentration level of 1.6 mg/L (nominal).
- In order to calculate the mean exposure concentrations, where the measured concentration was not detected, the concentration was taken as the Limit of Detection (LOD = 0.013 mg/L) (according to OECD 23; paragraph 3.3). Therefore the corresponding measured geometric mean test material concentrations were: 0.03; 0.05; 0.08; 0.49 and 3.31 mg/L in presence of algae, while the measured geometric mean test material concentrations were: 0.063; 0.224; 0.53; 1.51 and 3.98 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, with algae present there is some loss of test material, particularly at lower concentrations; the loss was much greater in the presence of algae. 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 tested concentrations of 1.024, 0.256 and 0.64 mg/L (nominal) was not or not clearly reduced at the section days 2-3. Even if it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition, however decrease of the measured test material concentrations in absence of algae cannot be negligible and thus biological results are related to the measured geometric mean test material concentrations for the aquatic concentrations without algae present.

CELL NUMBERS
- The cell number in each flask was determined at the 24th, 48th, 72nd hours.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
- There were no morphological deviations observed during the experiment.

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 measured concentration range of 0.53 and 3.98 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.224 mg/L (measured).
- The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.63 mg/L (95 % confidence limits: 0.58 – 0.70 mg/L (measured).

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 measured concentration range of 0.53 and 3.98 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.224 mg/L (measured).
- The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.42 mg/L (95 % confidence limits: 0.39 – 0.46 mg/L (measured).

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 measured concentration range of 0.53 and 3.98 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.224 mg/L (measured).
- The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.43 mg/L (95 % confidence limits: 0.39 – 0.48 mg/L (measured).

Results with reference substance (positive control):

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 2007 for Windows software.
- 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 71.50 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 11.81 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.20 %.

- 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 [mg/L]	Measured [mg/L]	0-24 h		0-48 h		0-72 h
		µ	%	µ	%	µ	%
Control	0.00	0.0620	0.0	0.0610	0.0	0.0593	0.0
0.1024	0.063	0.0640	-3.2	0.0613	-0.4	0.0591	0.4
0.256	0.224	0.0538	13.2	0.0598	2.1	0.0590	0.5
0.64	0.53	0.0345*	44.3	0.0332*	45.5	0.0429*	27.6
1.6	1.51	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0
4.0	3.98	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

*: 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 [mg/L]	Measured [mg/L]	0-24 h		0-48 h		0-72 h
		A	%	A	%	A	%
Control	0.00	42.0	0.0	298.0	0.0	1358.0	0.0
0.1024	0.063	44.0	-4.8	304.0	-2.0	1356.0	0.4
0.256	0.224	32.0	23.8	264.0	11.4	1292.0	4.9
0.64	0.53	16.0*	61.9	80.0*	73.2	384.0*	71.7
1.6	1.51	0.0*	100.0	0.0*	100.0	0.0*	100.0
4.0	3.98	0.0*	100.0	0.0*	100.0	0.0*	100.0

*: 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 [mg/L]	Measured [mg/L]	0-72 h
		Y	%
Control	0.00	70.5	0.0
0.1024	0.063	69.3	1.7
0.256	0.224	69.0	2.1
0.64	0.53	21.3*	69.7
1.6	1.51	0.0*	100.0
4.0	3.98	0.0*	100.0

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

Results of the Analysis

Measured Concentrations in Absence of Algae Cells with the 95 % Confidence Intervals

Nominal Conc. mg/L	Measured concentrations at the start mg/L	Measured concentrations after 1 day mg/L	Measured concentrations after 2 days mg/L	Measured concentrations at the end mg/L
Control	n.d.	n.d.	n.d.	n.d.
0.1024	0.0900 ± 0.0269	0.0889 ± 0.011	0.0593 ± 0.0102	0.0329 ± 0176
0.256	0.279 ± 0.039	0.220 ± 0.033	0.250 ± 0.024	0.165 ± 0.158
0.64	0.612 ± 0.061	0.608 ± 0.037	0.492 ± 0.071	0.439 ± 0.040
1.6	1.81 ± 0.32	1.63 ± 0.08	1.54 ± 0.04	1.15 ± 0.51
4.0	4.31 ± 0.51	4.24 ± 0.46	3.93 ± 0.45	3.51 ± 1.86

n.d. = Not detected

 

Measured Concentrations in Presence of Algae Cells with the 95 % Confidence Intervals

Nominal Conc. mg/L	Measured concentrations after 1 day mg/L	Measured concentrations after 2 days mg/L	Measured concentrations at the end mg/L
Control	n.d.	n.d.	n.d.
0.1024	0.0349 ± 0.0070	n.d.	n.d.
0.256	0.159 ± 0.030	n.d.	n.d.
0.64	0.464 ± 0.035	n.d.	n.d.
1.6	1.73 ± 0.02	1.36 ± 0.66	n.d.
4.0	4.30 ± 0.36	3.57 ± 1.19	1.81 ± 0.69

n.d. = Not detected

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 0-72 hour average specific growth rates, areas and yield were significantly different from that of the control group in the measured concentration range of 0.53 – 3.98 mg/L. The geometric mean measured EC50 values were 0.63, 0.43 and 0.42 mg/L for growth rate, yield and biomass, respectively. The overall NOEC was determined as 0.224 mg/L (measured); the overall LOEC was determined as 0.53 mg/L (measured).

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 2.5 and one untreated control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 0.1024, 0.256, 0.64, 1.6 and 4.0 mg/L.

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 olution 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: 0.03; 0.05; 0.08; 0.49 and 3.31 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 0.063; 0.224; 0.53; 1.51 and 3.98 mg/L for the nominal concentrations of 0.1024, 0.256, 0.64, 1.6 and 4.0 mg/L, respectively.

Based on these results, with algae present there is some loss of test material, particularly at lower concentrations; the loss was much greater in the presence of algae. Most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

Even if, 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, however decrease of the measured test material concentrations in absence of algae cannot be negligible and thus biological results are related to the measured geometric mean test material concentrations for the aquatic concentrations without algae present.

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 hour average specific growth rates, areas and yield were significantly different from that of the control group in the measured concentration range of 0.53 – 3.98 mg/L. The geometric mean measured EC50 values were 0.63, 0.43 and 0.42 mg/L for growth rate, yield and biomass, respectively. The overall NOEC was determined as 0.224 mg/L (measured); the overall LOEC was determined as 0.53 mg/L (measured).

Description of key information

Under the conditions of this study, the 0-72 hour average specific growth rates, areas and yield were significantly different from that of the control group in the measured concentration range of 0.53 – 3.98 mg/L. The geometric mean measured EC50 values were 0.63, 0.43 and 0.42 mg/L for growth rate, yield and biomass, respectively. The overall NOEC was determined as 0.224 mg/L (measured); the overall LOEC was determined as 0.53 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.63 mg/L

EC10 or NOEC for freshwater algae:

0.224 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 2.5 and one untreated control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 0.1024, 0.256, 0.64, 1.6 and 4.0 mg/L.

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: 0.03; 0.05; 0.08; 0.49 and 3.31 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 0.063; 0.224; 0.53; 1.51 and 3.98 mg/L for the nominal concentrations of 0.1024, 0.256, 0.64, 1.6 and 4.0 mg/L, respectively.

Based on these results, with algae present there is some loss of test material, particularly at lower concentrations; the loss was much greater in the presence of algae. Most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

Even if, 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, however decrease of the measured test material concentrations in absence of algae cannot be negligible and thus biological results are related to the measured geometric mean test material concentrations for the aquatic concentrations without algae present.

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 hour average specific growth rates, areas and yield were significantly different from that of the control group in the measured concentration range of 0.53 – 3.98 mg/L. The geometric mean measured EC50 values were 0.63, 0.43 and 0.42 mg/L for growth rate, yield and biomass, respectively. The overall NOEC was determined as 0.224 mg/L (measured); the overall LOEC was determined as 0.53 mg/L (measured).