(S)-N,N'-dibutyl-2-[(1-oxododecyl)amino]glutaramide

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:

30 January 2018 to 02 February 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:

- Duplicate samples were taken (2 x ~2 mL) in plastic tubes from the control and at the applied test concentration levels (each replicate) at the beginning of the test and 24 hour intervals thereafter during the experiment.
- After sampling, samples were frozen and kept approximately at -20 °C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set is retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).

Vehicle:

no

Details on test solutions:

- Because the test material is very poorly soluble in water, a test 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 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 % saturated solution.
- Note that the test material dissolved very slowly, hence although the duration of formulation procedure is relatively long for a molecule which in not highly stable, it was necessary to use a long saturation solution method to achieve maximal concentrations.
- 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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:

22.6 and 22.9 °C

pH:

7.22 – 8.33

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.
The corresponding calculated test material concentrations were: 0.20, 0.26, 0.35, 0.47 and 0.63 μg/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 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 2.0
- 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: Because slight inhibition was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment.

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 – t1) + [(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 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits not determined.

Remarks:

Calculation based on the nominal concentrations

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Calculation based on the nominal concentrations

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.47 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Calculation based on the nominal concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.63 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits: 0.58 - 0.69 µg/L

Remarks:

Calculation based on the nominal concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 0.55 - 0.66 µg/L

Remarks:

Calculation based on the nominal concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.26 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

and yield

Remarks on result:

other: Calculation based on the nominal concentrations

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

and yield

Remarks on result:

other: Calculation based on the nominal concentrations

Details on results:

CONCENTRATIONS OF THE TEST MATERIAL
- Due to the very low solubility of the test material extremely low test material concentrations needed to be measured during the test. In case of the lowest test concentration (6.25 % saturated solution) the measured concentration was already below the Limit of Quantification (LOQ) at the start of the experiment. At the nominal levels of 12.5 and 25.0 % saturated solutions measured concentration was determined at the start of the experiment, but was below the Limit of Quantification 24 hours later.
- In case of the two highest test material levels of 50.0 and 100.0 % saturated solution test material could be measured at the start of the test and 24 hours later, but it could not be determined later during the experiment. At these levels, in order to calculate mean value, where measured concentration was detected, but not quantified, concentration was taken as the half of the Limit of Quantification (LOQ = 0.48 μg/L) according to OECD 23; paragraph 3.3.
- In case of the three lowest concentration levels (where analytical concentrations could not be determined) exposure concentrations were extrapolated from calculated value of the next concentration level using the same mean separation factor (1.3404) between the measured geometric mean concentrations at the two highest levels.
- The corresponding calculated test material concentrations were: 0.20, 0.26, 0.35, 0.47 and 0.63 μg/L. Biological results are related both to the nominal and calculated test material concentrations.

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 any observed morphological deviations 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 at the concentration range of 50.0 – 100.0 % saturated solution (nominal), accordingly the No Observed Effect Concentration (NOEC) was determined as 25.0 % saturated solution (nominal).
- The 72 h ErC50 value could not be calculated and determined to be higher than 100.0 % saturated solution.

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 at the concentration range of 25.0 - 100.0 % saturated solution (nominal), accordingly the No Observed Effect Concentration (NOEC) was determined as 12.5 % saturated solution (nominal).
- The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 89.50 % saturated solution (95 % confidence limits: 71.13 – 112.63 % saturated solution).

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 at the tested concentration range of 25.0 - 100.0 % saturated solution (nominal), accordingly the No Observed Effect Concentration (NOEC) determined as 12.5 % saturated solution (nominal).
- The 72 h EyC50 value was determined to be higher than 100.0 % saturated solution, [101.99 % saturated solution was calculated (95 % confidence limits: 82.71 – 125.75 % saturated solution) by Probit analysis (TOXSTAT software)].

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 EyC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EbC 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 (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50 of the test material could not be calculated and therefore determined directly from the raw data (due to the slight inhibition observed). The EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.
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.

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

Nominal Concentration (% sat. sol.)	Growth rate (μ) and % inhibition of μ
	0-24 h		0-48 h		0-72 h
	µ	%	µ	%	µ	%
Control	0.0589	0.0	0.0620	0.0	0.0591	0.0
6.25	0.0569	3.4	0.0613+	1.2	0.0589	0.2
12.5	0.0569	3.4	0.0602+	3.0	0.0590	0.1
25.0	0.0529	10.2	0.0544*	12.2	0.0583	1.4
50.0	0.0529	10.2	0.0505*	18.6	0.0561*	5.1
100.0	0.0498	15.4	0.0401*	35.3	0.0490*	17.1

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

+: At these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

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

Nominal Concentration (% sat. sol.)	Area under the Growth Curves (A) and Percentage Inhibition of A
	0-24 h		0-48 h		0-72 h
	A	%	A	%	A	%
Control	38.0	0.0	300.0	0.0	1356.0	0.0
6.25	36.0	5.3	288.0	4.0	1328.0	2.1
12.5	36.0	5.3	276.0	8.0	1308.0	3.5
25.0	32.0	15.8	216.0*	28.0	1152.0*	15.0
50.0	32.0	15.8	188.0*	37.3	980.0*	27.7
100.0	28.0	26.3	128.0*	57.3	596.0*	56.0

*: 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

Nominal Concentration (% sat. sol.)	Yield (Y) and % inhibition of Y
	0-72 h
	Y	%
Control	69.3	0.0
6.25	68.7	1.0
12.5	69.0	0.5
25.0	65.3*	5.8
50.0	55.7*	19.7
100.0	33.0*	52.4

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

Validity

- The cell density in the control cultures increased by the factor of 70.33 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 13.67 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.55 %.

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 50.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 25.0 % saturated solution (nominal) and LOEC was determined as 50.0 % saturated solution (nominal). Furthermore, areas and yield were significantly different also from that of the control group in the examined concentration range of 25.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 12.5 % saturated solution (nominal) and LOEC was determined as 25.0 % saturated solution (nominal). The EC50 values were >100, >100 and 89.50 % saturated solutions 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 test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions.

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 material during the exposure period. The corresponding calculated test material concentrations were: 0.20, 0.26, 0.35, 0.47 and 0.63 μg/L.

The biological results are based on both of the nominal and calculated test material concentrations.

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

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

The ErC50 of the test material could not be calculated and therefore determined directly from the raw data (due to the slight inhibition observed). The 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 were significantly different from that of the control group in the examined concentration range of 50.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 25.0 % saturated solution (nominal) and LOEC was determined as 50.0 % saturated solution (nominal). Furthermore, areas and yield were significantly different also from that of the control group in the examined concentration range of 25.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 12.5 % saturated solution (nominal) and LOEC was determined as 25.0 % saturated solution (nominal). The EC50 value for yield was calculated as 0.63 µg/L (0.58 – 0.69 µg/L). The EC50 value for biomass was calculated as 0.60 µg/L (0.55 – 0.66 µg/L). NOEC was calculated as 0.26 µg/L and the LOEC was calculated as 0.35 µg/L.

The EC50 value for growth rate was calculated as > 0.63 µg/L. The 95 % confidence limits were not determined. The NOEC was calculated as 0.35 µg/L and the LOEC was calculated as 0.47 µg/L.

Description of key information

Under the conditions of this study, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 50.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 25.0 % saturated solution (nominal) and LOEC was determined as 50.0 % saturated solution (nominal). Furthermore, areas and yield were significantly different also from that of the control group in the examined concentration range of 25.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 12.5 % saturated solution (nominal) and LOEC was determined as 25.0 % saturated solution (nominal). The EC50 values were >100, >100 and 89.50 % saturated solutions for growth rate, yield and biomass, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.35 µg/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 test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions.

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 material during the exposure period. The corresponding calculated test material concentrations were: 0.20, 0.26, 0.35, 0.47 and 0.63 μg/L.

The biological results are based on both of the nominal and calculated test material concentrations.

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

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

The ErC50 of the test material could not be calculated and therefore determined directly from the raw data (due to the slight inhibition observed). The 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 were significantly different from that of the control group in the examined concentration range of 50.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 25.0 % saturated solution (nominal) and LOEC was determined as 50.0 % saturated solution (nominal). Furthermore, areas and yield were significantly different also from that of the control group in the examined concentration range of 25.0 - 100.0 % saturated solution (nominal), therefore the NOEC was determined as 12.5 % saturated solution (nominal) and LOEC was determined as 25.0 % saturated solution (nominal).The EC50 value for yield was calculated as 0.63 µg/L (0.58 – 0.69 µg/L). The EC50 value for biomass was calculated as 0.60 µg/L (0.55 – 0.66 µg/L). NOEC was calculated as 0.26 µg/L and the LOEC was calculated as 0.35 µg/L.

The EC50 value for growth rate was calculated as > 0.63µg/L. The 95 % confidence limits were not determined. The NOEC was calculated as 0.35µg/L and the LOEC was calculated as 0.47µg/L.