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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:
08 March 2013 to 06 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD and EU test guidelines in an accreditied GLP laboratory
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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)
Analytical monitoring:
yes
Details on sampling:
Analysis of the Test Item ConcentrationsFor measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (with algae). At the same sampling times, duplicate samples were also taken from the control.For the aged samples taken at test end, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions.Immediately after sampling, 100 mL ethyl acetate was added to each sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (-20 °C).The concentrations of the test item were determined in one of the duplicate test medium samples from all test concentrations at the start and end of the test.From the control samples, one of the duplicate samples was analyzed per sampling time.
Vehicle:
yes
Details on test solutions:
Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 569.9 mg of the test item in 5700 mL of test water in a 5 L Erlenmeyer flask with a glass stopper (closed system). This preparation was supported by intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
Test type:
static
Water media type:
other: Reconstituted test water (AAP Medium)
Remarks:
See "Any other information on materials and methods incl. tables" for more details.
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not specified
Hardness:
Not specified
Test temperature:
Temperature-controlled water bath at a temperature of 22 °C
pH:
7.9 to 8.4
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
The following treatments were tested: dilutions 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate with the loading rate of 100 mg/L. This corresponds to theoretical concentrations of 4.5, 10, 21.7, 45.5 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.
Details on test conditions:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 6500 Lux (range: 5980 to 6950 Lux, measured at nine places in the experimental area). The light intensity was within a ±15% deviation from the average light intensity as recommended by the guideline.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 66 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence interval 62-70 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Remarks on result:
other: 95% confidence interval 26-37 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 40 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 36-44 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 16 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Remarks on result:
other: 95% confidence interval 11-20mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 37-35 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Remarks on result:
other: 95% confidence interval 6.8-15 mg/L
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Details on results:
At the start and end of the test, the analytically determined concentrations of 2,2-dimethylpropane-1,3-diy 2-ethylhexanoate (CAS: 28510-23-8) in the test media were below the LOQ of 0.002 mg/L. Therefore, the biological results were related to the theoretical test item concentration of 4.5, 10, 21.7, 45.5 and 100 mg/L.The test item had significant inhibitory effect on the growth rate (μ) of the algae after the test period of 72 hours at the test concentration of 10 mg/L and at all higher test concentrations in addition to this the yield (Y) of the algae was statistically significantly reduced at the same test concentrations. The 72-hour NOEC based on the growth rate (μ) was determined to be 4.5 mg/L, since at this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control. The NOEC and ECX -values for growth rate are summarized in the conclusion. Additionally, the corresponding values for yield are given. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:2.2 and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.In the control, the biomass increased by a factor of 153 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 18%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period with the exception of turbidity observed in the dilution 1:2.2 (Day 3) and the undiluted filtrate (Day 2 and 3). The pH increased in the control from 8.1 at test start to 8.4 at test end fulfilling the requirement of the OECD guideline. The pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.9 to 8.4 during the test period. The water temperature during the test was maintained at 22 °C.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.93 mg/L (Harlan Study Number D64298), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71-1.7 mg/L).
Reported statistics and error estimates:
The test item had a significant inhibitory effect on the growth rate (μ) of the algae after the test periods of 72 hours at the test concentration of 10 mg/L and at all higher test concentrations (results of Welch t-test, one-sided smaller, α = 0.05, Table 2).The yield (Y) of the algae was statistically significantly reduced at the same test concentration.The 72-hour NOEC based on the growth rate (μ) was determined to be 4.5 mg/L, since at this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control.

At the start and end of the test, the analytically determined concentrations of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) in the test media were below the LOQ of 0.002 mg/L (see analytical results and Table 4 in Appendix 1). Therefore, the biological results were related to the theoretical test item concentrations of 4.5, 10, 21.7, 45.5 and 100 mg/L.

 

The test item had a significant inhibitory effect on the growth rate (μ) of the algae after the test periods of 72 hours at the test concentration of 10 mg/L and at all higher test concentrations (results of Welch t-test, one-sided smaller, α = 0.05).

 

The yield (Y) of the algae was statistically significantly reduced at the same test concentration.

 

The 72-hour NOEC based on the growth rate (μ) was determined to be 4.5 mg/L, since at this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control.

 

The NOEC and ECX-values for growth rate are summarized in the conclusion. Additionally, the corresponding values for yield are given.

 

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:2.2 and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

 

In the control, the biomass increased by a factor of 153 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control during 72 hours was 18%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

 

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period with the exception of turbidity observed in the dilution 1:2.2 (Day 3) and the undiluted filtrate (Day 2 and 3).

 

The pH increased in the control from 8.1 at test start to 8.4 at test end fulfilling the requirement of the OECD guideline. The pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.9 to 8.4 during the test period. The water temperature during the test was maintained at 22 °C.

 

Validity criteria fulfilled:
yes
Executive summary:

The impact of the test item 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

 

Due to the low solubility of the test item in test water, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2.2, 1:4.6, 1:10 and 1:22 were used as test media. This corresponds to theoretical concentrations of 100, 45.5, 21.7, 10 and 4.5 mg/L. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The test was performed as far as possible in a closed system, because the test item was determined to be volatile.

 

At the start and end of the test, the analytically determined concentrations of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) in the test media were below the LOQ of 0.002 mg/L. Therefore, the reported biological results were based on the theoretical test item concentrations.

 

The biological results of the study were as follows:after 72 h

Parameter

Growth rate

Yield

EC50[mg/L]

66

31

95% confidence interval

62-70

26-37

EC20[mg/L]

40

16

95% confidence interval

36-44

11-20

EC10[mg/L]

31

11

95% confidence interval

27-35

6.8-15

NOEC [mg/L]

4.5

4.5

LOEC [mg/L]

10

10

 

Description of key information

Toxicity to Pseudokirchneriella subcapitata in a 72-Hour Algal Growth Inhibition Test

The biological results of the study were as follows:

72h EC50 66 mg/L (Growth rate)

72h EC50 31 mg/L (Yield)

72h EC20 40 mg/L (Growth rate)

72h EC20 16 mg/L (Yield)

72h EC10 31 mg/L (Growth rate)

72h EC10 11 mg/L (Yield)

72h NOEC 4.5 mg/L (Growth rate)

72h NOEC 4.5 mg/L (Yield)

72h LOEC 10 mg/L (Growth rate)

72h LOEC 10 mg/L (Yield)

The test substance 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) is therefore not classified accordance with CLP Regulation (EC) 1272/2008

Key value for chemical safety assessment

EC50 for freshwater algae:
31 mg/L
EC10 or NOEC for freshwater algae:
4.5 mg/L

Additional information

Toxicity to Pseudokirchneriella subcapitata in a 72-Hour Algal Growth Inhibition Test

The study was performed under consideration of the procedures indicated by the following internationally accepted guidelines and recommendations:

• OECD Guidelines for the Testing of Chemicals, No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test, 2006.

• Commission Regulation (EC) No 761/2009 C.3: Algal Inhibition Test, 2009.