Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2015 - 26 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed on the control at the beginning and at the end of the test, and on the samples containing the test material at the beginning of the tests and 24-hour intervals thereafter directly after sampling. Since the test item is evaporable and due the sensitivity of algae cells and sterile circumstances, the sampling after 24 and 48 hour were done from separate replicate vessels for analytical sampling. At the end of the test, the analytical sampling and the observation was performed from the same vessel. In all cases, determination of test substance concentrations was only performed on one replicate vessel at each test concentration from the additional sample without algae in order to distinguish degradation and adsorption of the test item to algae. The samples were analysed by an HPLC-UV method.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated test item stock solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) and stirred for about an hour using magnetic stirrer. Test solutions of lower test concentrations were prepared by appropriate diluting of this stock solution
- Evidence of undissolved material (e.g. precipitate, surface film, etc): the non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100 % saturated solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY
- Age of inoculum (at test initiation): four-day-old algal pre-culture
- Method of cultivation: the pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.8 – 23.2 °C
pH:
7.00 (at the start of the study) and 8.01 – 8.44 (at the end of the study)
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.1, 9.8, 31.3 and 100% saturated solutions.
Measured concentrations: 0.008; 0.045; 0.162; 0.604 and 2.202 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: closed bottle test.
- Type (delete if not applicable): closed.
- Initial cells density: 3.3 x 10E3 algal cells/mL test medium.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: yes. At the beginning of the test the pH was adjusted to 7.0 by addition of HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 8146 lux (equivalent to 110 µE/m2/s) with fluorescent lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell numbers were determined at 24, 48 and 72 hours after the treatment 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: 3.2
- Range finding study: two concentration range-finding tests were conducted. Algal cells were exposed for 72 h using the standard method in the first study and using closed vessels in the second study. Both test was performed with two replicates per test concentrations and three replicates in the untreated control. Taking into account that the test item is a highly volatile substance the test was performed with confirmatory analytical measurements at the start and at the end of the experiment in case of the second preliminary test in order to better define loss of the test substance during the exposure period.
- Test concentrations: 0, 0.1, 1.0, 10.0 and 100.0 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: because significant toxic effect was observed during the performed preliminary range-finding tests, five test concentrations in a geometric series (factor 3.2) and one control were tested in the definitive test. The following nominal concentrations were tested in the main test: 1.0, 3.1, 9.8, 31.3 and 100 % saturated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (02 – 05 March 2015).
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.045 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.:
0.462 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 0.374 – 0.570)
Results with reference substance (positive control):
The 72h ErC 50: 0.84 mg/L, (95 % confidence limits: 0.77 – 0.92 mg/L)
The 72h EbC 50: 0.55 mg/L, (95 % confidence limits: 0.50 – 0.60 mg/L)
The 72h EyC 50: 0.49 mg/L, (95 % confidence limits: 0.45 – 0.53 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 using Probit analysis by TOXSTAT software. 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. 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.

Endpoint values:

NOEC (72 h): 0.008 mg/L (based on growth rate)

NOEC (72 h): 0.008 mg/L (based on yield)

NOEC (72 h): 0.008 mg/L (based on biomass)

LOEC (72 h): 0.045 mg/L (based on growth rate)

LOEC (72 h): 0.045 mg/L (based on yield)

LOEC (72 h): 0.045 mg/L (based on biomass)

EC50 (72 h): 0.462 mg/L (based on growth rate)

EC50 (72 h): 0.129 mg/L (based on yield)

EC50 (72 h): 0.127 mg/L (based on biomass)

Table 1. Growth rates (µ) and Percentage Inhibition of µ during Test Period.

Concentration

Growth rate (µ) and % inhibition of µ

Nominal

Measured

0–24 h

0–48 h

0–72 h

[% sat. sol.]

[mg/L]

µ

%

µ

%

µ

%

Control

0.0

0.0863+

0.0

0.0767

0.0

0.0708

0.0

1.0

0.008

0.0807

6.5

0.0759+

1.0

0.0705

0.5

3.1

0.045

0.0751

13.0

0.0710*

7.5

0.0683*

3.6

9.8

0.162

0.0654+*

24.2

0.0602*

21.5

0.0620+*

12.5

31.3

0.604

0.0462*

46.5

0.0231*

69.9

0.0154*

78.3

100

2.202

0.0462*

46.5

0.0231*

69.9

0.0154*

78.3

*: 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.

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 0.045 – 2.202 mg/L (measured), correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.008 mg/L (measured).The 72 h ErC50value was determined [by Probit analysis (TOXSTAT software)] as 0.462 mg/L(95 % confidence limits: 0.374 – 0.570 mg/L)].

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

Concentration

Concentration

Nominal

Measured

0–24 h

0–48 h

0–72 h

[% sat. sol.]

[mg/L]

A

%

A

%

A

%

Control

0.0

28.0

0.0

210.1

0.0

1010.2

0.0

1.0

0.008

24.0

14.3

196.1

6.7

972.2

3.8

3.1

0.045

20.0

28.5

156.1*

25.7

812.2*

19.6

9.8

0.162

16.0*

42.8

100.1*

52.4

508.2*

49.7

31.3

0.604

8.0*

71.3

24.1*

88.5

40.2*

96.0

100

2.202

8.0*

71.3

24.1*

88.5

40.2*

96.0

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

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 whole examined concentration range of 0.045 – 2.202 mg/L (measured), correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.008 mg/L (measured).The 72 h EbC50value was determined [by Probit analysis(TOXSTAT software)] as0.127 mg/L (95 % confidence limits: 0.104 – 0.156 mg/L).]

Table 3. Yeld (Y) and Percentage Inhibition of Y during the Test Period.

Concentration

Concentration

Nominal

Measured

0–72 h

[% sat. sol.]

[mg/L]

Y

%

Control

0.0

53.8

0.0

1.0

0.008

52.3

2.8

3.1

0.045

45.0*

16.4

9.8

0.162

28.3*

47.4

31.3

0.604

0.7*

98.8

100

2.202

0.7*

98.8

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

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 whole examined concentration range of 0.045 – 2.202 mg/L (measured), correspondingly the No Observed Effect Concentration (NOEC) was determined as 0.008 mg/L (measured).The 72 h EyC50value was determined [by Probit analysis (TOXSTAT software)] as 0.129 mg/L (95 % confidence limits: 0.108 – 0.155 mg/L)].

Table 4. Calculation of exposure concentrations (in presence of algae)

Nominal
concentration

(% sat. sol.)

Measured concentrations
(mg/L)

Geometric mean

start

24h

48h

72h/end

mg/L

Control

n.d.

-

-

n.d.

-

1.0

0.0416

0.0129

0.0025*

0.0025*

0.0076

3.1

0.126

0.0475

0.0313

0.0209

0.0445

9.8

0.414

0.178

0.120

0.0780

0.1621

31.3

1.35

0.631

0.459

0.340

0.6038

100

4.46

2.45

1.72

1.25

2.2016

n.d. – not detected

*   concentration was below the Limit of Quantification. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.005 mg/L)according to OECD 23; paragraph 3.3.

Table 5. Calculation of exposure concentrations (in absence of algae)

Nominal
concentration

(% sat. sol.)

Measured concentrations
(mg/L)

Geometric mean

start

24h

48h

72h/end

mg/L

Control

n.d.

-

-

-

-

1.0

0.0416

0.0126

0.0025*

0.0025*

0.0076

3.1

0.126

0.0471

0.0320

0.0230

0.0457

9.8

0.414

0.189

0.128

0.0884

0.1725

31.3

1.35

0.610

0.439

0.353

0.5977

100

4.46

2.27

1.58

1.26

2.1188

n.d. – not detected

*   concentration was below the Limit of Quantification. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.005 mg/L)according to OECD 23; paragraph 3.3

In case of the lowest tested concentration level the measured concentration was below the Limit of Quantification (LOQ) 48 hours after the treatment and at the end of the test both in presence and absence of algae. Inorder to calculate the mean exposure concentration, where measured concentration was detected, but not quantified, concentration was taken as the half of the Limit of Quantification (LOQ = 0.005 mg/L) according to OECD 23; paragraph 3.3.(see Tables 6 - 7.).

 

The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item.

Validity criteria fulfilled:
yes
Remarks:
(Cell density increased by a factor of 54.17 (> 16-fold growth). Mean coefficient of variation for section-by-section growth rate was 21.80 (<35%). Coefficient of variation of mean specific growth rates was 0.62 (<7%)).
Conclusions:
The EC50 value based on growth rate was determined to be 0.462 mg/L (measured). The overall NOEC was determined as 0.008 mg/L measured), the overall LOEC was determined as 0.045 mg/L (measured).
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green algae Pseudokirchneriella subcapitata over an exposure period of 72 hours in a closed system. The test was performed in accordance with the OECD Guideline 201 and following the Principles of GLP. Based on two performed preliminary range-finding tests, five test concentrations in a geometric series (factor 3.2) and once control were tested in the main experiment. The nominal concentrations used in the main experiment were 1.0, 3.1, 9.8, 31.3 and 100% saturated solutions. The test item was measured at the start of the experiment and in 24 -hour intervals thereafter. The corresponding measured geometric mean test item concentrations were: 0.008, 0.045, 0.162, 0.604 and 2.202 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item. With respect to the inhibitory effect of the test item, the 0 -72 h average specific growth rates, yield and areas were significantly different from that of the control group in the examined concentration range of 0.045 -2.202 mg/L (measured). The EC50 value based on growth rate was determined to be 0.462 mg/L. The overall NOEC was determined as 0.008 mg/L measured), the overall LOEC was determined as 0.045 mg/L (measured).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance 7-chloro-3-methyl-1-benzothiophene (SER-4) which shares the aryl functional group chlorophenyl with the substance 1-[(4-chlorophenyl)sulfanyl]propan-2-one (SER-3) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.508 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.009 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
Based on the available information for the read-across approach, the 72h-EC50 of the test item in algae is 0.508 mg/L and the NOEC is 0.009 mg/L.

Description of key information

Read-across from analogue:

Key study: Test method according to OECD Guideline 201. GLP study. The EC50 value based on growth rate was determined to be 0.462 mg/L (measured). The overall NOEC was determined as 0.008 mg/L measured), the overall LOEC was determined as 0.045 mg/L (measured). Based on the available information for the read-across approach, the 72h-EC50 of the test item in algae is 0.508 mg/L and the NOEC is 0.009 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.51 mg/L
EC10 or NOEC for freshwater algae:
0.009 mg/L

Additional information

Read-across from analogue: Key study: The effect of the test item was assessed on algal growth using the unicellular green algae Pseudokirchneriella subcapitata over an exposure period of 72 hours in a closed system. The test was performed in accordance with the OECD Guideline 201 (GLP study). Based on the results of the preliminary range-finding tests, the nominal concentrations used in the main experiment were 1.0, 3.1, 9.8, 31.3 and 100% saturated solutions. The corresponding measured geometric mean test item concentrations were: 0.008, 0.045, 0.162, 0.604 and 2.202 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item. The EC50 value based on growth rate was determined to be 0.462 mg/L. The overall NOEC was determined as 0.008 mg/L measured), the overall LOEC was determined as 0.045 mg/L (measured). Based on the available information for the read-across approach, the 72h-EC50 of the test item in algae is 0.508 mg/L and the NOEC is 0.009 mg/L.