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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 - 31 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: samples were taken from the 4th replicate; 1 mL was sampled and directly analysed
- Sample storage conditions before analysis: samples were stored in a freezer at approx. -25 °C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: appropriate dilution series of a stock solution (160 mg/L in 1 L of growth medium); stirred for 1.5 h at room temperature; All stock solutions and the medium were prepared with purified water processed using an ELGA PURELAB system.
- Controls: yes, test item control
- Eluate: no
- Differential loading: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen
- Age of inoculum (at test initiation): Three days old pre-culture was used for the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.0 - 23.5 °C
pH:
7.66 - 8.98 (control)
7.34 - 8.66 (test item concetrations)
Nominal and measured concentrations:
Control; 10; 20; 40; 80 and 160 mg test item/L (nominal)
Control, 4.96, 12.5, 32.2, 75.7 and 157 mg test item/L (mean measured concentration)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type (delete if not applicable): open (covered with air-permeable silicone-sponge caps)
- Material, size, headspace, fill volume: 250 mL conical glass flasks and were oscillated by continuously stirring on a laboratory shaker with 125 rpm
- Initial cells density: 10000 cells/mL
- Control end cells density: 297388 cells/mL (mean)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium according to the guideline OECD 201; stock solutions and the medium were prepared with purified water processed using an ELGA ‘PURELAB’ system.
- details on growth medium are summarized in table 1 within section " Any other information on material and methods incl tables"
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH of the medium is obtained at equilibrium with air to the range of 7.5 to 8.0
- Photoperiod: continuously
- Light intensity and quality: 7478 - 7561 lux OSRAM “day light”; The light intensity was measured daily using an illuminance meter LI-189 (LI-COR, Lincoln, USA with radiation sensor) with a cosine (2pi) receptor in lux on a level with the surface of the test media.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell numbers were determined during the test (24, 48, 72 h) using an electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany); At test end the cell number of the three highest test concentrations was determined by microscopically counting due to a functional disorder of the electronic cell counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: non-GLP range-finder test with DMSO and 0.1, 1.0, and 10.0 mg/L
- Results used to determine the conditions for the definitive study: no significant inhibition of growth rate and yield compared to the controls up to 10 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
37.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidencial limits
Remarks:
34.1 - 41.4 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidencial limits
Remarks:
10.5 - 15.9 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: see remarks
Remarks:
inhibition was 7.1 % at 12.5 mg/L. The statistical NOEC was calculated to be <4.96 mg/L (2.9 % inhibition) due to the low variability of the replicates. Since inhibition below 10 % is generally not considered to be ecotoxicologically relevant, the NOEC was set at 12.5 mg/L. It is suggested to use the EC10 values for the risk assessment, as recommended in the OECD guideline 201.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidential limits
Remarks:
13.5 - 17.4 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.43 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidential limits
Remarks:
4.62 - 7.91 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 4.96 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): normal appearances of the algae cells despite increasing cell debris with increasing inhibition
Results with reference substance (positive control):
The nominal ErC50 value of 2.42 mg/L (95% confidence limits 1.95 - 3.03 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
The test results showed an inhibition with levels around 50 % and were statistically analysed to determine EC10, EC20 and EC50 values for growth rate [r] and yield [y] together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values by using a computer programme, e.g. ToxRat. The NOEC and LOEC values were determined using determined by the Williams Multiple Sequential t-test. The computer program ToxRat Professional was be used for statistical evaluations.

Table 1: Analytical Results: Analysed test item (CAS 141-17-3) and DEGBE concentrations (CW in units of mg/L) and 'percent of nominal' values

 

CAS 141-17-3

CAS 141-17-3

CAS 141-17-3

DEGBE

Sampling

Nominal test item conc.

Measured analyte conc., CW

Percent of nominal

Measured analyte conc., CW

 

[mg/L]

[mg/L]

[%]

[mg/L]

0 h

Control-

<LOQ

-

<LOQ

 

10

8.98

89.8

<LOQ

 

20

18.1

90.7

<LOQ

 

40

36.9

92.3

<LOQ

 

80

76.1

95.1

<LOQ

 

160

158

98.5

<LOQ

2 h

Control-

<LOQ

-

<LOQ

 

10

9.30

93.0

<LOQ

 

20

19.1

95.5

<LOQ

 

40

38.6

96.5

<LOQ

 

80

78.3

97.8

<LOQ

 

160

156

97.4

<LOQ

6 h

Control-

<LOQ

-

<LOQ

 

10

9.57

95.7

<LOQ

 

20

18.4

92.1

<LOQ

 

40

39.1

97.7

<LOQ

 

80

80.4

101

<LOQ

 

160

162

102

<LOQ

24 h

Control-

<LOQ

-

<LOQ

 

10

8.91

89.1

0.52

 

20

18.6

92.9

<LOQ

 

40

38.4

95.9

<LOQ

 

80

80.3

100

<LOQ

 

160

157

98.3

<LOQ

48 h

Control-

<LOQ

-

<LOQ

 

10

5.71

57.1

1.77

 

20

12.5

62.3

2.71

 

40

31.3

78.3

3.72

 

80

76.2

95.2

4.08

 

160

160

100

5.76

72 h

Control-

<LOQ

-

<LOQ

 

10

0.37

3.65

3.66

 

20

2.55

12.7

6.47

 

40

16.8

41.9

8.33

 

80

64.3

80.3

6.93

 

160

150

93.5

6.84

 

 

Mean measured

 

 

0 – 72 h

Control-

<LOQ

-

 

10

4.96

 

 

 

20

12.5

 

 

 

40

32.2

 

 

 

80

75.7

 

 

 

160

157

 

 

 

Table 2: Biological Results: Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

Mean measured concentration [mg/L]

% Inhibition of growth rate

% Inhibition of yield

Control

0.00

0.00

4.96

2.90

15.2

12.5

7.10

32.9

32.2

41.7

90.9

75.7

85.4

99.6

157.1

86.3

99.6

 

Table 3: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

297

yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

13.8%

yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

1.2%

yes

 

Validity criteria fulfilled:
yes
Remarks:
Please refer to table 3 in "Any other information on results incl. tables"
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
30 Jul 2010 - 16 Sep 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Analytical monitoring:
yes
Details on sampling:
Range-finding test
Samples were taken from the test preparations for chemical analysis at 0 and 72 hours for stability determination. All samples were stored at approx. -20 °C prior to analysis.
Definitive test
Samples were taken from control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis, if necessary. Given a significant decline in measured test concentrations over the 72-hour test period, additional test samples were prepared in the definitive test with the omission of the algal cells in order to determine whether the decline observed was due to instability or adsorption of the test item by the algal cells.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Range finding
A 100 mg sample of the test item was dissolved directly in the culture medium and the volume adjusted to 1 liter giving a 100 mg/L stock solution.These were serially diluted to provide additional stock solutions of 10, 1.0 and 0.10 mg/L. A 500 mL aliquot of each of these stock solutions was separately inoculated with algal suspensions (7.6 mL) to give the required test concentrations of 0.10, 10 and 100 mg/L.

Definitive test
Samples of 100 and 32 mg of the test item were separately dissolved directly in culture medium and the volumes adjusted to 1 liter giving stocksolutions of 100 and 32 mg/mL, respectively. These were serially diluted to provide additional stock solutions of 10, 3.2 and 1.0 mg/L. A 500 mL aliquot of each of these stock solutions was separately inoculated with algal suspension (4.9 mL) to give the test concentrations of 1.0, 3.2, 32 and 100 mg/L.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Name: Desmodesmus subspicatus
- Strain: CCAP 276/20- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyl, Scotland
- Age of inoculum (at test initiation): No information
- Method of cultivation: master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 +/- 1 °C. Prior tothe start of the test, sufficient master culture was added to approximately 100 ml volumes of culture media in conical flasks to give an initial cell density of approximately 1000 cells/mL. Flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 +/- 1 °C until the algal cell density was approximately 10,000 to 100,000 cells/mL. The culture medium used for both the range-finding and definitive tests was that same as that used to maintain the stock culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 +/- 1 °C
pH:
pH values increased from 7.4 - 7.7 at 0 hours to 7.8 to 8.0 at 72 h. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and was therefore within the guideline limits for this study.
Nominal and measured concentrations:
The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg/L. The geometric mean measured test concentrations based on measurements at 0 hours and 72 hours were 0.10, 0.18, 0.33, 0.55 and 66 mg/L, respectively, for the 1.0, 3.2, 10, 32 and 100 mg/L nominal concentrations. Expressed as a percentage, this corresponds to 10, 6, 3, 2 and 66%, respectively, for the 1.0, 3.2, 10, 32 and 100 mg/L nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:- Type: 250 mL conical flasks, containing 100 mL of test preparation
- Initial cells density: 4 x 10 E+03 cells/mL (approx.)
- Control end cells density: 1.16 x 10 E+05 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: approx. 7000 lux (warm light, 380 - 730 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell densities were determined using a Coulter(R) Multisizer Particle Counter after 0, 24, 48 and 72 h.
Reference substance (positive control):
yes
Remarks:
(potassium dichromate; study conducted between 21 June 2010 and 2 July 2010)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. interval: 20 - 28 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. interval: 0.45 - 0.52 mg/ L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.33 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.:
0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
5.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Remarks on result:
other: 95% conf. interval: 8.9 - 14 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Remarks on result:
other: 95% conf. interval: 0.31 - 0.39 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: biomass (increased yield)
Details on results:
Range finding test
The results indicated no effect on growth at the nominal test concentrations of 0.1 and 1.0 mg/L. However, growth was observed to be reduced at the10 and 100 mg/L nominal concentrations. Thus, test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were chosen for the definitive testing.Definitive test
Mean growth rate and yield values for the definitive test are given in Table 1.
Validation criteria: Cell concentrations of control cultures increased by a factor of 29 after 72 hours. This satisfies the OECD Guideline that states enhancement must beat least a factor of 16 after 72 hours.
- Mean cell density of control at 0 hours: 4.07 x 10^3 cells/ml- Mean cell density of control at 72 hours: 1.16 x 10^5 cells/mlThe mean coefficients of variation for section by section specific growth rate for the control cultures was 20%, satisfying the validation criteria(OECD Guideline) which states the mean must not exceed 35%. The coefficients of variation for average specific growth rate for the control over the test period was 4% and hence satisfied the validation criteriongiven in the OECD guideline stating that this value must not exceed 7%.
Results with reference substance (positive control):
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following: ErC50 (0-72 hours): 0.74 mg/L
EyC50 (0-72 hours): 0.37 mg/L
The results from the positive control were within the normal ranges for this material.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Biological Results

Table 1: Growth Rate and Yield, Definitive Test (mean values, +/- SD)

Nominal Conc. (mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 - 72 hours

% inhibition

0 - 72 hours

% inhibition

Control

0.047 +/- 0.002

-

1.12E+05 +/- 1.40E+04

-

1.0

0.046 +/- 0.002

2

1.08E+05 +/- 1.18E+04

4

3.2

0.045 +/- 0.001

3

1.01E+05 +/- 5.63E+03

10

10

0.039 +/- 0.001

16

6.36E+04 +/- 6.70E+03

43

32

0.017 +/- 0.01

64

1.19E+04 +/- 8.64E+03

89

100

0.01 +/- 0.004

98

2.45E+02 +/- 1.20E+03

100

Given measured losses of test chemical concentration in the range finding study, a control in the definitive test was prepared with the omission of algal cells. Analysis of these test preparations at 72 hours showed a similar decline in concentrations. This suggested that loss of test chemical concentration was due to instability rather than adsorption to algal cells.

A worst-case analysis of the data dictated the use of geometric mean measured concentrations based on the 0 hour and 72 hour analyses. In cases where measured concentrations were below the limit of quantitation (LOQ) for the analytical method, a value of one-half or the LOQ was employed to enable calculation of the geometric mean.

Table 2: Analytical Results:

Concentration of Adipic Acid, bis[2-(2-butoxyethoxy)ethyl] Ester in Aqueous Test Media Samples Determined Using GC Analysis

Sample

Nominal Concentration (mg/L)

Concentration Found (mg/L) *

Expressed as a Percentage of the Nominal Concentration (%)*

0 hours

Control

<LOQ

-

 

1.0

0.941

94

 

3.2

3.05

95

 

10

9.95

100

 

32

27.9

87

 

100

87.8

88

72 hours

Control

<LOQ

-

 

1.0

<LOQ

-

 

3.2

<LOQ

-

 

10

<LOQ

-

 

32

<LOQ

-

 

100

49.7 50

 

72 hours

Control

<LOQ

-

No algae

1.0

<LOQ

-

 

3.2

<LOQ

-

 

10

<LOQ

-

 

32

<LOQ

-

 

100

55.3

55

 

Table 3: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

29

yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

20%

yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

4%

yes

 

Validity criteria fulfilled:
yes
Remarks:
Please refer to table 3 in "Any other information on results incl. tables"
Conclusions:
The test item caused an effect on Desmodesmus subspicatus over the 72-hour study period. Reduced growth rates and biomass increases wererecorded with EC50 values of 23 and 11 mg/L, respectively, based on nominal test item concentrations. When expressed as geometric meanmeasured concentrations, EC50 values of 0.48 and 0.35 mg/L, respectively, were reported based on growth rate and biomass increases.

Description of key information

ErC50 (72 h): 37.6 mg/L (OECD 201, growth rate, based on mean measured concentration)

ErC10 (72 h): 13.3 mg/L (OECD 201, growth rate, based on mean measured concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:
37.6 mg/L
EC10 or NOEC for freshwater algae:
13.3 mg/L

Additional information

Two experimental studies are available investigating the toxicity of the substance Bis(2-(2-butoxyethoxy)ethyl) adipate (CAS 141-17-3) to aquatic freshwater algae in accordance with OECD 201 (GLP).

The key study was performed with the freshwater algae Pseudokirchneriella subcapitata. Exposure concentrations were in the range of 10, 20, 40, 80 and 160 mg /L (nominal) corresponding to mean measured concentrations of 4.96, 12.5, 32.2, 75.7 and 157 mg /L. The concentrations of the test item (CAS 141-17-3) and the degradation product 2-(2-butoxyethoxy)ethanol (DEGBE, CAS 112-34-5) were assessed by chemical analysis using LC-MS/MS in all test solutions sampled at the beginning of the test and after an incubation period of 2, 6, 24, 48, and 72 hours. The test item concentrations ranged between 89.1 and 102% of nominal during the first 24 hour test period. After 72 h the measured values were between 3.65 and 93.5% of the nominal values. Only minor amounts of the degradation product DEGBE were formed after 72 h and were between 3.66 and 8.33 mg/L. The mean measured concentrations of the test item were used for the evaluation, since the concentrations decreased more than 20% compared to the initial values in the three lowest treatment. For the relevant measurement parameter growth rate the ErC50 (72 h) and ErC10 (72 h) were calculated to be 37.6 mg/L and 13.3 mg/L (mean measured). A NOErC (72 h) of 12.5 mg/L was derived for growth rate. For yield the EyC50 (72 h) and EyC10 (72 h) was calculated to be 15.3 mg/L and 6.43 mg/L (mean measured), respectively. The NOEyC was estimated to be below 4.96 mg/L.

The supporting study was performed with Desmodesmus subspicatus and exposed to the test item concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 72 h. Analytical measurements at test start showed measured test concentrations to range from 87% to 100% of nominal. Analysis of the test preparations after 72 hours showed a decline in measured test concentrations in the range of less than the Iimit of quantification (LOQ) of the analytical method employed (which was determined to be 0.022 mg/L) to 50% of nominal. Based on these results the toxicological endpoints were evaluated based on the nominal concentrations and the geometric mean measured test concentrations. Based on the nominal test item concentrations the ErC50 (72 h) was determined to be 23 mg/L for growth rate. The NOErC (72 h) based on the nominal concentrations was 10 mg/L for growth rate. Based on the geometric mean measured concentrations the EC50 was 0.48 mg/L for growth rate. The NOEC was observed to be 0.33 mg/L for growth rate.

In comparison to the key study the supporting study showed a high decrease of test item concentration after 72 h. The measured concentrations (geometric mean) dropped to ≤ 10% of nominal at the concentrations relevant for calculation of effect concentrations (ErC10, ErC50). In contrast the nominal concentrations in the key study were maintained to a higher extent (49.6 - 98.1% of nominal). Thus, the effect concentrations derived from the key study give a more realistic estimate of toxicity to aquatic algae. The lower effect concentrations in the supporting study are expected to highly overestimate the toxicity to aquatic algae.