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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant, guideline study, no restrictions, available as an unpublished report, fully adequate for assessment, tested with the source substance CAS 68201-60-5. Based on the structural similarities to one of the components of the target substance (the tested substance is the sodium salt), this study is considered valid for read-across.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the temperature in the main study was higher than stated in the study plan; the cell start concentration was 5.5*10^3/mL instead of 2-5*10^3/mL as stated in the study plan; - the real concentration in treatment 46 mg/L was 48 mg/L
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
same as above
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material
PHYSICO-CHEMICAL PROPERTIES
- Melting point: gradually softened to a viscous liquid, but it was not possible to determine a melting point.
- Boiling point: decomposes from approximately 119°C
- Vapour pressure: 0.000097 Pa at 25°C.
- Water solubility (under test conditions): At a loading rate of 10 g/L the solubility, expressed as percentage of loading rate, was ca. 50% w/w, and at a loading rate of 1 g/L was ca. 47% w/w at pH 8.1 – 9.6
- log Pow: 3.42 – >6.5
Analytical monitoring:
yes
Details on sampling:
The samples, which were collected at t=0 h, were measured after storage in a fridge for one day.

Vehicle:
no
Details on test solutions:
- Before the experiment, water-accommodated fractions (WAF) of the concentrations to be tested were prepared. This was done by weighing the nominal load, adding the appropriate amount of deionised water and shaking for 24 hours. After membrane filtration, the solutions were used to prepare the treatments. Since the solutions were diluted by algal pre-culture and nutrient medium, 1.25 times the nominal amount was used in preparation of the WAFs.
For each treatment, 160 mL of the respective test solution (1.25-fold concentrated) was prepared and mixed with 40 mL of the test culture.
- Control: deionised water with nutrient medium and alga
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus (unicellular freshwater green alga)
- Strain: CHODAT
- Source (laboratory, culture collection): MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)

ACCLIMATION
- Acclimation period: 72 hours
- Culturing media and conditions (same as test or not): Before the start of the test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 72 hours. Before usage, the culture was checked on the absence of cell aggregates. Cell density was measured and adjusted by addition of algal medium. The adjusted pre-culture was mixed with the same amount of 10-fold-nutrient solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Range from 25 to 23 °C
pH:
At the start of the test: range from 7.4 to 7.8
At the end of the test: range from 7.2 to 9.3
Nominal and measured concentrations:
Nominal concentrations: 0, 4.6, 10, 22, 46, 100 mg/L of the test item, corresponding to 0, 2.3, 5, 11, 23, 50 mg/L as 100% resin acids and rosin acids, maleated, sodium salts
Measured concentrations: 0, -, -, 16, 36, 75 mg/L of the test item, corresponding to 0, -, -, 8, 18, 37.5 mg/L as 100% resin acids and rosin acids, maleated, sodium salts
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks of 50 mL were incubated open on an orbital shaker
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six
- Reference study: No. of vessels per positive control (replicates): Three for each treatment;
No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: Continuous light
- Light intensity and quality: The light intensity was 5800 lux.
- Analitical determination: The measured concentrations after 72 hours were in a range between 76% and 143% of the start concentration and between 66% and 115% of the nominal concentration, respectively. Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations. Moreover, as the measured DOC values of the nominal treatments 4.6 mg/L and 10 mg/L were in the same range as the blank control values, these values were not used for further calculations.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined by microscope counts.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50 % aqueous solution
Basis for effect:
other: growth rate, AUC and yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: 100 % resin acids and rosin acids, maleated, sodium salts
Basis for effect:
other: growth rate, AUC and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50 % aqueous solution
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 37.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
100 % resin acids and rosin acids, maleated, sodium salts
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50 % aqueous solution
Basis for effect:
other: AUC
Remarks on result:
other: 22-65 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
100 % resin acids and rosin acids, maleated, sodium salts
Basis for effect:
other: AUC
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50 % aqueous solution
Basis for effect:
other: Yield
Remarks on result:
other: 25-47 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
100 % resin acids and rosin acids, maleated, sodium salts
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Microscopical observations at the end of the test: normal growth was observed in the control and in the treatments at 4.6, 10, 22 mg/L (nominal concentrations), less growth was observed in the treatments at 46 and 100 mg/L (nominal concentrations).
- As inhibition values in the two lowest treatments (4.6 mg/L and 10 mg/L nominal concentration) were higher than in the next higher treatment (22 mg/L nominal concentration) where no significant inhibition was observed, the two lowest treatments were not used for evaluation of the biological results.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h-EC50 for the growth rate endpoint: 1.40 mg/L (95%-confidence-interval: > 1.1 mg/L)
- 72h-EC50 for the AUC (Area Under Curve) endpoint: 0.61 mg/L (95%-confidence-interval: 0.51 - 0.74 mg/L)
- 72h-EC50 for the Yield endpoint: 0.38 mg/L (95%-confidence-interval: 0.30 - 0.47 mg/L)
- 72h-NOEC: 0.25 mg/L
- Other: In the microscopical observations, no cells were observed at the highest treatment concentrations of 1.0 and 1.6 mg/L

Reported statistics and error estimates:
For the treatments with the nominal test item concentrations 22 and 46 mg/L (measured concentrations are 16 and 36 mg/L, respectively), it was tested whether the differences between treatment and control were significant. The values for the AUC, Yield and the growth rate were used for the assessment of observed effect levels. In order to select a suitable test for significance, it was checked whether equality of variance was given, using the following equation: F= s1^2/s2^2, with s1 being the greater variance and s2 the smaller one. The calculated value F was compared with the F-test table (level of significance 95%). If the calculated value was smaller than the tabular value, equality of variance was given, and therefore the t-test was used. With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance.
Validity criteria fulfilled:
yes
Conclusions:
The following effects of Resin acids and Rosin acids, maleated, sodium salts on the test system were observed under the conditions reported (concentration range 4.6 - 100 mg/L, 50 % aqueous solution, 72 h exposition, 23-25 °C): a NOEC of 16 mg/L (measured concentration) and a LOEC of 36 mg/L (measured concentration) were observed for all three endpoints (growth rate, AUC, Yield), corresponding to 8 mg/L and 18 mg/L, respectively, of 100 % Resin acids and Rosin acids, maleated, sodium salts. The EC50s for the growth rate, AUC, and Yield were stated to be: > 75 mg/L, 41 mg/L and 35 mg/L (measured concentrations, 50 % aqueous solution), corresponding to 37.5, 20.5 and 17.5 mg/L, respectively, of 100 % Resin acids and Rosin acids, maleated, sodium salts.
Executive summary:

The 72-h algal growth inhibiton study, performed with Resin acids and Rosin acids, maleated, sodium salts, was conducted according to the following guidelines: OECD 201 and EU C.3. All validity criteria were met. One main study was performed using five concentrations ranging from 4.6 to 100 mg/L. As the measured DOC values of the treatments with the nominal concentrations 4.6 mg/L and 10 mg/L were in the same range as the blank control values, these values were not used for further calculations. The measured concentrations after 72 hours were in a range between 76% and 86% of the start concentration and between 66% and 69% of the nominal concentration, respectively. Therefore, the determination of the biological results was based on the geometrical mean of the measured concentrations. The EC50s of the positive control, i.e. potassium dichromate, were tested in a current reference test. The values laid within the normal range of the laboratory. The following deviations from the study plan were documented: 1) the temperature in the main study was higher than stated in the study plan; 2) the cell start concentration was 5.5*10^3/mL instead of 2 -5*10^3/mL as stated in the study plan. As all validity criteria were fulfilled and normal growth in the controls were observed both deviations were stated as uncritical. 3) Moreover, the real concentration in treatment 46 mg/L was 48 mg/L and therefore more than±1 mg/L deviation. This was stated uncritical because the biological results were based on the measured concentration.

This is a GLP-compliant and guideline study. Therefore, the results are reliable without restriction.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant, guideline study, available as an unpublished report, fully adequate for assessment, tested with the source substance CAS 91722-01-9. Based on the structural similarities to one of the components of the target substance (the tested substance is the calcium salt), this study is considered valid for read-across.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the temperature during the main study was higher than stated in the study plan. As normal growth in the controls was observed, this was stated as uncritical
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material
PHYSICO-CHEMICAL PROPERTIES
- Melting point: fuse together from 120°C
- Boiling point: decomposes at 313°C.
- Vapour pressure: 0.0000022 Pa at 25°C
- Water solubility (under test conditions): 0.0158 g/L - 0.0558 g/L
- log Pow: 4.69 - 5.84 at ca. pH 2
Analytical monitoring:
yes
Details on sampling:
- Sampling method: on day 0, samples were collected, filtrated, and stored at 2 - 8 °C until measurement. On day 3, samples were measured directly after filtration.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: as solubility lies below 100 mg/L, the water-accomodated fractions were prepared for the test. This was done by weighing the nominal loadings, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solutions were filtrated through 0.45 µm filters. For each treatmenr, 160 mL of the respective test solution (1.25-fold concentrated) was prepared and mixed with 40 mL of the test culture.
- Differential loading: 10, 18, 32, 56, 100 mg/L
- Control: deionised water with nutrient medium and alga
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Desmodesmus subspicatus (unicellular freshwater green alga)
- Strain: CHODAT
- Source (laboratory, culture collection): MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)

ACCLIMATION
- The algae were kept as stock culture on solid agar at 8 °C. Four days before the start of the test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 96 hours. The resulting culture grew exponentially.
- Any deformed or abnormal cells observed: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
25 - 27 °C
pH:
Ranged from 7.5 (at the end of the test) to 8.1 (at the start of the test)
Nominal and measured concentrations:
Nominal concentrations: 0, 10, 18, 32, 56, 100 mg/L
Measured DOC concentrations in the treatment and in the controls were in the same range and therefore results are based on nominal loadings
Details on test conditions:
TEST SYSTEM
- Glass flasks of 50 mL were used as test vessels. The test vessels were filled with 45 mL ± 5 mL of the mixture and were incubated open for 72 hours shaken on an orbital shaker.
- Initial cells density: 2206 cell number/mL (mean value)
- Control end cells density: 133831 cell number/mL (mean value)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not performed
- Light intensity and quality: Continuous at 4800 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : From the cell numbers, the Growth Rate µ (at 24, 48, 72 h), the Area Under the Curve AUC (at 24, 48, 72 h) and the Yield were calculated. NOEC, EL50, and LOEC values for these endpoint were determined.
- Determination of cell concentrations: Photometric measurement of optical density (Abs 440 nm)
- Other: At the end of the test, the treatments were observed microscopically in order to assess the appearance of the alga and detect abnormalities.

TEST CONCENTRATIONS
- Test concentrations: 10, 18, 32, 56, 100 mg/L (nominal concentrations)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fraction
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fraction
Basis for effect:
other: AUC (Biomass integral)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fraction
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fraction
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fraction
Basis for effect:
other: AUC (Biomass integral)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Conc. based on:
other: water accommodated fraction
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none.
- Normal cells were microscopically observed at the end of the test at 0, 10, 18, 32, 56, 100 mg/L (nominal concentrations).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 72 h (Growth rate µ): 1.40 mg/L (95%-confidence interval: > 1.1 mg/L)
- EC50 72 h (AUC): 0.61 mg/L (95% confidence interval: 0.51-0.74 mg/L)
- EC50 72 h (Yield): 0.38 mg/L (95% confidence interval: 0.30-0.47 mg/L)
- NOEC 72 h: 0.25 mg/L
- LOEC 72 h: 0.4 mg/L
Reported statistics and error estimates:
For the treatment with the test item concentration 100 mg/L, it was tested whether the differences between treatment and control were significant. The values for the area under the curve, Yield and the growth rate were used for the assessment of observed effect levels. In order to select a suitable test for significance, it was checked whether equality of variance was given. Equality of variance was tested using the following equation: F= s1^2/s2^2, with s1 being the greater variance and s2 the smaller one. The calculated value F was compared with the F-test table (level of significance 95%). If the calculated value was smaller than the tabular value, equality of variance was given. As equality of variance was given, the t-test was used.
With the t-test it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with grade of freedom: n1+n2-2, level of significance 95%).
Determination of the NOEC: for all endpoint µ, AUC, Yield, the differences between the treatment and the control can be considered as not significant (level of significance: 97.5%) as the calculated t-values were smaller than the limit of significance. Therefore, the respective NOECs are stated as being equal or higher than 100 mg/L.
Determination of the LOEC: for all endpoints µ, AUC and Yield, the differences between the treament 100 mg/L and the control can be considered as not significant (level of significance: 97.5%) as the calculated t-values were smaller than the limit of significance. All other treatments showed no inhibition compared to the control. Therefore, the respective LOECs are stated as being higher than 100 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
No significant inhibition of algal growth was observed in any treatment and in the controls. Therefore, the NOELs of the test item for the endpoints µ, AUC and Yield were stated as being higher than 100 mg/L. The LOELs for the test item are stated as being higher than 100 mg/L nominal load for all endpoints. Correspondly, the EL50s for the test item for the same endpoints were above the concentration of 100 mg/L.
Executive summary:

This study was conducted in accordance with the OECD Guideline No. 201-2006, and with the EU Method C.3. One valid main study was performed, where all validity criteria were met. The measured DOC concentrations in the blank control were in the same range as in the treatment. The determination of the biological results was based on the nominal loadings. The EC50s of the positive control were tested in a current reference test. For the estimation of the EC50s of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory. The following deviation from the study plan was documented: the temperature during the main study was higher than stated in the study plan; as normal growth in the controls was observed, this was stated as uncritical. GLP-compliant study, therefore the results are reliable without restrictions.

Description of key information

- EC50 (72 h) >37.5 mg/L (OECD 201; Resin acids and Rosin acids, maleated, sodium salts)
- EL50 (72 h) >100 mg/L (OECD 201; Resin acids and Rosin acids, maleated, calcium salts)

The NOEC of the substance is set at the water solubility level in a worst case approach (1.38 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.38 mg/L
EC10 or NOEC for freshwater algae:
1.38 mg/L

Additional information

Aquatic toxicity to invertebrates was assessed by using studies with metal salts of Rosin, maleated. The results considered to be representative for the UVCB (see document on read-across). The EC50 were all above the water solubility level. The difference in the results between the two salts must be attributed to the difference in solubility.

Aquatic toxicity of Resin Acids and Rosin Acids, maleated, Calcium Salts on algae was tested in a study conducted in accordance with the OECD Guideline 201 and EU Method C.3 (LAUS, 2012f). No significant inhibition of algal growth was observed in any treatment and in the controls. Therefore, the NOELs of the test item for the endpoints µ, AUC and Yield were stated as being higher than 100 mg/L. The LOELs for the test item are stated as being higher than 100 mg/L nominal load for all endpoints. The EL50s for the test item for the same endpoints were above the concentration of 100 mg/L.

In a similar study, Resin acids and Rosin acids, maleated, sodium salts was tested and following effects on the test system were observed under the conditions reported (concentration range 4.6 - 100 mg/L, 50 % aqueous solution, 72 h exposition, 23-25 °C): a NOEC of 16 mg/L (measured concentration) and a LOEC of 36 mg/L (measured concentration) were observed for all three endpoints (growth rate, AUC, Yield), corresponding to 8 mg/L and 18 mg/L, respectively, of 100 % Resin acids and Rosin acids, maleated, sodium salts. The EC50s for the growth rate, AUC, and Yield were stated to be: > 75 mg/L, 41 mg/L and 35 mg/L (measured concentrations, 50 % aqueous solution), corresponding to 37.5, 20.5 and 17.5 mg/L, respectively, of 100 % Resin acids and Rosin acids, maleated, sodium salts.