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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:
19 March 2018 to 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex IV Part C, C.3.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
other: Japanese Guidelines
Version / remarks:
JMAFF 2-7-3. The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.
Qualifier:
according to
Guideline:
other: Japanese Guidelines
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).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
The following nominal concentrations were tested: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions. Test concentrations were analytically determined at the start of the experiment and 24-hour intervals thereafter in order to better define loss of the test substance during the exposure period.
Vehicle:
no
Details on test solutions:
Because the Test Item was poorly soluble in water, a test solution were 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 Item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of Test Item into the test medium (OECD Medium) 2 days before the start of the test. This solution was shaken for approximately 24 hours at approximately 30°C and then was equilibrated for approximately 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.
The test solutions were prepared by appropriate diluting of this stock solution and these solutions were distributed into the appropriate test flasks.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 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.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: 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:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not specified
Hardness:
Not specified
Test temperature:
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 temperature was 22.6-22.8°C measured in the flask and were in the range of 21.7 and 23.0 °C measured within the climate chamber.
pH:
The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.17 – 8.31 during the experiment.
Dissolved oxygen:
Not specified
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
The concentrations in the definitive test are based on the results of the preliminary range-finding test and were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions.
Details on test conditions:
PERFORMANCE OF THE TEST
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
Due to a lack of stability data for the 72 hours exposure period we intend to extend the study to provide more data to allow a better interpretation.
Additional samplings for analysis at 24 hour intervals were used in order to better define the possible loss of the test substance during the exposure period.
The test was performed with 5 replicates per test concentration and 8 replicates in the control group. In the test concentrations 3 replicates were used for observations and 2 replicates for the 24 and 48 hour analytical measurements. In the control 6 replicates were used for observation and 2 replicates for the 24 hours and 48 hour analytical measurements.
Volumes of 100 mL test solution per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range-Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the Test Item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
During the formulation procedure the stock solution was prepared by the similar method described above (3.2.1.) except the time of equilibration which was about 24 hours. The test solutions were prepared by appropriate dilution of this stock solution.

Test Item concentrations in the Definitive Test
Because toxic response was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.3) and one control were tested in the main experiment.
The concentrations in the definitive test are based on the results of the preliminary range-finding test and were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions.

OBSERVATIONS
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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.005 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.002 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield & Biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.044 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.015 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
VALIDITY
The cell density in the control cultures increased by the factor of 69.67 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 18.55 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.85 %.
All validity criteria were met; therefore, the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM
The following nominal concentrations were tested: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions. Test concentrations were analytically determined at the start of the experiment and 24-hour intervals thereafter in order to better define loss of the test substance during the exposure period.
The Test Item was under the limit of quantification (The corresponding measured geometric mean and calculated Test Item concentrations were: 0.0021, 0.00487, 0.01126, 0.02959 and 0.05911 mg/L. The biological results are based on the measured and calculated concentrations.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There was no morphological deviation observed in the 3 lowest concentration levels. Chuff cells were observed at the highest concentration level at 48h and 72h observation, in addition chuff cells were also observed at the concentration level of 0.02959 mg/L during the study at the 72 hour observation occasion.

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 concentrations of 0.01126, 0.002959 and 0.05911 mg/L (measured) correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.00487 mg/L (extrapolated).
The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.0442 mg/L (95 % confidence limits: 0.0365 – 0.0536 mg/L).

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 concentrations of 0.01126, 0.002959 and 0.05911 mg/L (measured) and in 0.00487 mg/L (extrapolated), accordingly the No Observed Effect Concentration (NOEC) determined as 0.0021 mg/L (extrapolated).
The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.017 mg/L (95 % confidence limits: 0.0147 – 0.0196 mg/L).

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 concentrations 0.01126, 0.002959 and 0.05911 mg/L (measured) and in 0.00487 mg/L (extrapolated), accordingly the No Observed Effect Concentration (NOEC) determined as 0.0021 mg/L (extrapolated).
The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.0153mg/L (95 % confidence limits: 0.0134 – 0.0173mg/L).
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)
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 Item were determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample 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 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Calculation of the exposure concentrations

Nominal concentrations(% saturated solution)

Measured concentrations(mg/L)

Geometric mean(mg/L)

Extrapolated value(mg/L)

0h

24h

48h

72h

Control

0.54

1.23

2.84

6.52

15

n.d.

0.0143¥

0.0369 ¥

0.0852 ¥

0.189

0.435

-

0.00427 ¥

0.0100 ¥

0.0240 ¥

0.0632

0.118

-

<LOQ ¥

0.00228 ¥

0.00538 ¥

0.0136 ¥

0.0247 ¥

-

n.d. ¥

<LOQ ¥

0.00146 ¥

0.00472 ¥

0.00868 ¥

--

--

--

0.01126

0.02959

0.05911

--

0.0021

0.00487

--

--

--

n.d.: not detected

¥: Concentrated samples using the SPE (10 fold), therefore, the LOD is compliance to 0.00037 µg/mL and LOQ is compliance to 0.001 µg/mL after the concentration of samples.

 

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

Concentrations

Growth rate µ and % inhibition of µ

Nominal

(% saturated solution)

Measured/ Calculated geometric mean(mg/L)

0-24 h

0-48 h

0-72 h

µ

%

µ

%

µ

%

Control

0.54

1.23

2.84

6.52

15

--

0.0021

0.00487

0.01126

0.02959

0.05911

0.0569

0.0498

0.0498

0.0538

0.0458

0.0498

0.0

12.5

12.5

5.5

19.5

12.5

0.0622+

0.0613

0.0606

0.0549*

0.0373*

0.0269*

0.0

1.3

2.5

11.6

39.9

56.7

0.0589

0.0588

0.0571

0.0549*

0.0327*

0.0263*

0.0

0.2

3.2

6.9

44.5

55.4

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

+: at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the value calculated with EXCEL.

 

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

Concentrations

Growth rate A and % inhibition of A

Nominal

(% saturated solution)

Measured/ Calculated geometric mean(mg/L)

0-24 h

0-48 h

0-72 h

A

%

A

%

A

%

Control

0.54

1.23

2.84

6.52

15

--

0.0021

0.00487

0.01126

0.02959

0.05911

36.0

28.0

28.0

32.0

24.0

28.0

0.0

22.2

22.2

11.1

33.3

22.2

298.0

272.0

264.0

220.0*

108.0*

88.0*

0.0

8.7

11.4

26.2

63.8

70.5

1348.0

1304.0

1192.0*

988.0*

284.0*

188.0*

0.0

3.3

11.6

26.7

78.9

86.1

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

 

Yield

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

Concentration

Yield Y and % inhibition of Y

Nominal

(% saturated solution)

Measured/ Calculated geometric mean(mg/L)

0-72 h

Y

%

Control

0.54

1.23

2.84

6.52

15

--

0.0021

0.00487

0.01126

0.02959

0.05911

68.7

68.0

60.0*

51.0*

9.7*

5.7*

0.0

1.0

12.6

25.7

85.9

91.7

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

 

TEST CONDITIONS

pH-Values in the Test media at the Start and End of the Test

Concentration

pH-values

Nominal

Measured/ Calculated

Start

End

% sat. sol.

mg/L

Control

0.0

7.28

8.27

8.31

8.24

8.27

8.30

8.25

0.54

0.0021

7.22

7.96

8.20

8.16

1.23

0.00487

7.26

8.18

8.20

8.21

2.84

0.01126

7.17

8.14

8.10

8.06

6.52

0.02959

7.24

7.97

7.90

7.92

15

0.05911

7.22

7.26

7.28

7.24

 

Temperature in the Climate Chamber and in the Test Media during the Test

Parameter

Exposure Time

Day 0

Day 1

Day 2

Day 3

Temperature (°C) measured in flask

22.7

22.7

22.8

22.6

Min/max temperature (°C) of the climate chamber

Min

21.7

21.9

22.0

22.1

Max

23.0

23.0

22.9

22.8

 

Cell Number (x 104cell/mL) determined in the Main Experiment

Concentration

 

Number of cells

Nominal

Measured/ Calculated

0 h

24 h

48 h

72 h

% sat. sol.

mg/L

Control

0.0

 

 

 

 

 

 

Mean

SD

1

1

1

1

1

1

1.00

0.0

3

5

5

4

3

4

4.00

0.9

21

22

18

20

17

21

19.83

1.9

70

73

67

69

67

72

69.67

2.5

0.54

0.0021

 

 

 

Mean

SD

1

1

1

1.00

0.0

3

4

3

3.33

0.6

20

18

19

19.00

1.0

71

70

66

69.00

2.6

1.23

0.00487

 

 

 

Mean

SD

1

1

1

1.00

0.0

3

3

4

3.33

0.6

18

18

19

18.33

0.6

62

56

65

61.00

4.6

2.84

0.01126

 

 

 

Mean

SD

1

1

1

1.00

0.0

4

3

4

3.67

0.6

15

14

13

14.00

1.0

54

52

50

52.00

2.0

6.52

0.02959

 

 

 

Mean

SD

1

1

1

1.00

0.0

3

3

3

3.00

0.0

6

6

6

6.00

0.0

9

10

13

10.67

2.1

15

0.05911

 

 

 

Mean

SD

1

1

1

1.00

0.0

3

4

3

3.33

0.6

4

3

4

3.67

0.6

7

6

7

6.67

0.6

 

Validity criteria fulfilled:
yes
Conclusions:
Growth Rate [mg/L]
EC50: 0.0442
NOEC: 0.00487

Yield [mg/L]
EC50: 0.0153
NOEC: 0.0021

Biomass [mg/L]
EC50: 0.017
NOEC: 0.0021
Executive summary:

The effect of Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

 

Toxic response was observed during the preliminary range-finding test, therefore 5 test concentrations in a geometric series with a separation factor of 2.0 and 1 untreated Control were tested in the Main experiment.

 

The following nominal concentrations were tested: 0.54, 1.23, 2.84, 6.52 and 15% saturated solutions. Test concentrations were analytically determined at the start of the experiment and 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period. The corresponding measured geometric mean and calculated Test Item concentrations were: 0.0021, 0.00487, 0.01126, 0.02959 and 0.05911 mg/L. The biological results are based on the measured and calculated concentrations.

 

The test design included 5 replicates at each Test Item concentration and 8 replicates for the untreated Control.

 

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 Item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

 

In conclusion, under the conditions of this study, the calculated endpoints for the effect of the Test Item were the following:

Parameter

(0-72 h)

Growth rate (r)

[mg/L]

Yield (y)

[mg/L]

Biomass (b)

[mg/L]

EC50

95% conf. limits

0.0442

0.0365 – 0.0536

0.0153

0.0134 – 0.0173

0.017

0.0147 – 0.0196

NOEC

LOEC

0.00487

0.01126

0.0021

0.00487

0.0021

0.00487

 

Description of key information

The calculated endpoints for the effect of the Test Item were the following:

Parameter

(0-72 h)

Growth rate (r)

[mg/L]

Yield (y)

[mg/L]

Biomass (b)

[mg/L]

EC50

95% conf. limits

0.0442

0.0365 – 0.0536

0.0153

0.0134 – 0.0173

0.017

0.0147 – 0.0196

NOEC

LOEC

0.00487

0.01126

0.0021

0.00487

0.0021

0.00487

 

Key value for chemical safety assessment

EC50 for freshwater algae:
0.015 mg/L
EC10 or NOEC for freshwater algae:
0.005 mg/L

Additional information

The effect of Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

 

Toxic response was observed during the preliminary range-finding test, therefore 5 test concentrations in a geometric series with a separation factor of 2.0 and 1 untreated Control were tested in the Main experiment.

 

The following nominal concentrations were tested: 0.54, 1.23, 2.84, 6.52 and 15% saturated solutions. Test concentrations were analytically determined at the start of the experiment and 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period. The corresponding measured geometric mean and calculated Test Item concentrations were: 0.0021, 0.00487, 0.01126, 0.02959 and 0.05911 mg/L. The biological results are based on the measured and calculated concentrations.

 

The test design included 5 replicates at each Test Item concentration and 8 replicates for the untreated Control.

 

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 Item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

 

In conclusion, under the conditions of this study, the calculated endpoints for the effect of the Test Item were the following:

Parameter

(0-72 h)

Growth rate (r)

[mg/L]

Yield (y)

[mg/L]

Biomass (b)

[mg/L]

EC50

95% conf. limits

0.0442

0.0365 – 0.0536

0.0153

0.0134 – 0.0173

0.017

0.0147 – 0.0196

NOEC

LOEC

0.00487

0.01126

0.0021

0.00487

0.0021

0.00487