Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

(data suggesting possible test item instability was considered to be due to analytical variability; see Appendix 2, attached)

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)

Version / remarks:

Commission Directive 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Verification of test concentrations took place at 0 and 72 hours.

Vehicle:

no

Details on test solutions:

TEST WATER
- The culture medium is described in Appendix 1 (attached).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST SPECIES
- Source: Culture Centre of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria, UK.
- Pre-culture: Sterile culture medium (see Appendix 1, attached) was inocuated from a master culture of Scenedesmus subspicatus and incubated under continuous illumination (approximately 7000 lux) and aeration at 21 °C to give an algal suspension in log phase growth characterised by absorbance of 0.943 at 665 nm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

24 °C

pH:

7.7 to 10.1 (see Table 2, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

PRELIMINARY TEST
- Nominal concentrations of 0.10, 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

METHOD OF PREPARATION
- Test material (200 mg) was dispersed in culture medium with the aid of ultrasonic disruption and the volume was adjusted to 1 L to give a 200 mg/L stock solution.
- Serial dilutions were made from the stock solution to give 100, 50, 25 and 12.5 mg/L stock solutions.
- An aliquot of each stock solution (500 mL) was mixed with 500 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L.

EXPOSURE CONDITIONS
- Test vessels: Conical flasks (250 mL) containing test solution (100 mL) were loosely stoppered to reduce evaporation. All flasks were incubated and shaken at approximately 100 rpm in an orbital shaker.
- Experimental design: Five test concentrations plus one control each prepared in triplicate.
- Method of initiation: Algal pre-culture (500 mL) was added to sterile nutrient medium (500 mL) containing appropriate quantities of test material.
- Photoperiod: Continuous (approximately 7000 lux).
- Medium renewal: None.
- The pH of each test and control culture was measured at 0 and 72 hours.
- Measurement of growth: Samples were taken at 0, 24, 48 and 72 hours and absorbance was measured at 665 nm using a Jenway 6100 spectrophotometer. Cell densities of the control cultures were determined at 0, 24, 48 and 72 hours by direct counting with the aid of a haemocytometer to confirm that absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.

COMPARISON OF AREAS UNDER GROWTH CURVES
- The area under the growth curve was taken to be an index of growth and was calculated using the equation given in the attached data evaluation document.
- Percentage inhibition of growth at each test concentration was calculated by comparing the area under the test curve with that under the control curve (see data evaluation document, attached).
- Percentage inhibition of (see Table 3, attached) were plotted against test concentration (see Figure 3, attached). A line was fitted by eye and the EbC50 (72 h) value with respect to the area under the growth curve was read from the graph.

COMPARISON OF GROWTH RATES
- The average maximum growth rate for each culture was calculated from the straight section of the growth curve (see Figure 2, attached) using the equation shown in the attached data evaluation document.
- Percentage reductions in growth rate and the ErC50 (24-48 h) were calculated ( see data evaluation document, attached).

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: ErC50

Remarks:

value quoted as 24-48 h

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

PRELIMINARY TEST
- Results showed significant impairment of growth at 100 mg/L thereby indicating the test concentrations employed in the definitive test.

VALIDITY CRITERIA
- Mean cell density at 0 hours was determined to be 2.17 x 10E04 cells/mL.
- Mean cell density at 72 hours was determined to be 5.98 x 10E05 cells/mL.
- These data show that the cell concentration of the control cultures increased by a factor of 28 during the test.
- The result is in line with the OECD guideline which states that the enhancement must be at least a factor of 16 after 72 hours.

OBSERVATIONS
- No abnormalities were detected in any of the control or test cultures during visual inspection at 72 hours.
- pH values of the control and test cultures (see Table 2, attached) were observed to increase from 7.7 to 7.9 (0 h) to 9.0 to 10.1 (72 h). This effect was considered to be caused by the large number of algal cells in the log phase of growth (see Figure 2, attached) producing alkaline conditions during respiriation of oxygen and production of carbonates and bicarbonates as part of photosynthesis/respiration. The effect was not considered to affect the integrity of the study given that the cell densities of the cultures increased by a factor of 28 during the test.

VERIFICATION OF TEST CONCENTRATIONS
- Chemical analysis of the test preparations at 0 hours (see Appendix 2, attached) showed the measured concentrations were 90-100 % of nominal.
- Analysis of the test preparations at 72 hours showed the measured concentrations were 59-81 % of nominal.
- The decline in measured concentration was in line with stability analysis performed and was considered to be due to adsorption of the test material to the glassware and/or the algal cells in the test cultures. However, the data suggesting possible evidence of instability was considered to be due to analytical variability because the test material was shown to be stable in aqueous media in light and dark conditions when investigated in tests describing acute toxicity to rainbow trout and Daphnia magna (see Appendix 2, attached).
- In order to give a worst case analysis of the data it was considered justifiable to calculate the EC50 values based on the measured test concentrations at 72 hours.

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- One way analysis of variance was carried out on the area under the growth curve data for the control and all test concentrations after 72 hours.
- From the data given in Table 1 (attached) and presented graphically in Figure 1 (attached), it can be seen that absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.
- From the data given in Tables 2 and 3 (attached), it is clear that both the growth rate (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72 hour exposure period.
- From the data, EbC50 (72 h) was determined to be 64 mg/L and ErC50 (24-48 h) was determined to be 60 mg/L where Eb50 is the test concentration that reduced biomass by 50 % and ErC50 is the test concentration that reduced specific growth rate by 50 %.
- The area under the growth curve for the 6.25, 12.5 and 25 mg/L test concentrations was shown to be significantly greater (P < 0.05) than that for the control.
- The area under the growth curve for the 50 and 100 mg/L test concentrations was significantly less (P < 0.05) than the control.
- The data therefore indicates the No Observed Effect Concentration (NOEC) to be 25 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Chemical analysis showed that test item concentration declined from 90-100 % of nominal at 0 hours to 59-81 % of nominal at 72 hours. This effect was considered to be due to adsorption of the test material to the glassware and/or the algal cells in the test cultures and, to give a worst case analysis of the data, EC50 values were calculated based on the 72-hour measured concentrations. The EbC50 (72 h) value based on biomass was determined to be 45mg/L and the ErC50 (24-48 h) value based on growth rate was determined to be 42 mg/L. The No Observed Effect Concentration (NOEC) was reported as 15 mg/L.

Executive summary:

In a study performed according to OECD 201 and EU Method C.3 (Handley et al, 1994) the freshwater alga, Desmodesmus subspicatus (previous name: Scenedesmus subspicatus), was exposed to five nominal concentrations of test material plus mineral medium as the control for 72 hours under static conditions. Nominal concentrations of test item selected following a range-finding study were 6.25, 12.5, 25, 50 and 100 mg/L. Verification of test item concentration took place at 0 and 72 hours using a spectrophotometric method. Chemical analysis showed that test item concentration declined from 90-100 % of nominal at 0 hours to 59-81 % of nominal at 72 hours. This effect was considered to be due to adsorption of the test material to the glassware and/or the algal cells in the test cultures and, to give a worst case analysis of the data, EC50 values were calculated based on the 72-hour measured concentrations. The EbC50 (72 h) value based on biomass was determined to be 45mg/L and the ErC50 (24-48 h) value based on growth rate was determined to be 42 mg/L. The No Observed Effect Concentration (NOEC) was reported as 15 mg/L.

Description of key information

In a 72 -h study performed using Scenedesmus subspicatus the EbC50 (72 h) value based on biomass was determined to be 45mg/L and the ErC50 (24-48 h) value based on growth rate was determined to be 42 mg/L. The No Observed Effect Concentration (NOEC) was reported as 15 mg/L (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:

42 mg/L

EC10 or NOEC for freshwater algae:

15 mg/L

Additional information

In a key study performed according to OECD 201 and EU Method C.3 (Handley et al, 1994) the freshwater alga, Scenedesmus subspicatus (Chodat), was exposed to five nominal concentrations of test material plus mineral medium as the control for 72 hours under static conditions. Nominal concentrations of test item selected following a range-finding study were 6.25, 12.5, 25, 50 and 100 mg/L. Verification of test item concentration took place at 0 and 72 hours using a spectrophotometric method. Chemical analysis showed that test item concentration declined from 90-100 % of nominal at 0 hours to 59-81 % of nominal at 72 hours. This effect was considered to be due to adsorption of the test material to the glassware and/or the algal cells in the test cultures and, to give a worst case analysis of the data, EC50 values were calculated based on the 72-hour measured concentrations. The EbC50 (72 h) value based on biomass was determined to be 45mg/L and the ErC50 (24-48 h) value based on growth rate was determined to be 42 mg/L. The No Observed Effect Concentration (NOEC) was reported as 15 mg/L.