Castor oil, sulfated, ammonium salt

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

Study period:

From 2013, November 25 to 2014, January 8

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:

2006; Annex 5 corrected 28 July 2011.

Deviations:

yes

Remarks:

Deviations are listed below; The study integrity was not adversely affected by these deviations.

Principles of method if other than guideline:

List of protocol deviations
1. A correction factor of 1.41 was used for the purity/composition of the test substance.
Evaluation: Slightly higher concentrations than necessary were tested, but this did not affect the study outcome.
2. Combined limit/range-finding test: The vessels for the pre-culture have not been kept shaking during the first 2 days of incubation.
Evaluation: Algal growth in the pre-culture proved to be sufficient.
3. Final test: The test vessels of the highest test concentration were not pooled before the start of the recovery phase. Instead, all vessels have been tested individually.
Evaluation: This did not have an effect on the outcome of the recovery phase.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for Total Organic Carbon (TOC) analysis were taken from the control and all test concentrations without algae according to the schedule below.
Frequency at t=0 h, t=24 h and t=96 h
Volume 40 ml
Storage Samples were stored in a refrigerator until analysis.
At the end of the exposure period, the replicates without algae were pooled at each concentration before sampling.
Additionally, reserve samples of 40 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

Preparation of test solutions started with a nominal concentration of 141 mg/l containing 105 mg of the active ingredient (a.i.) per litre for the combined limit/range-finding test and with a nominal concentration of 212 mg/l containing 158 mg of the active ingredient (a.i.) per litre for the final test. These concentrations were magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 141 or 212 mg/l concentrations in test medium. The final test solutions were clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 23.1°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 24.0 and 24.8°C. Temperature remained within the limits prescribed by the protocol (22-26°C, constant within 2°C).

pH:

The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit).

Nominal and measured concentrations:

Four replicates of exponentially growing algal cultures were exposed to a control and to nominal test substance concentrations of 1.4, 4.9, 17, 61 and 212 mg/l, containing 1.0, 3.7, 13, 46 and 158 mg a.i. per litre

Details on test conditions:

The amount of test substance present in test medium was calculated from the measured amount of TOC in the test medium and the TOC content of the pure test substance.
A sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC) content (WIL Research Project 503334). TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose as total carbon (TC) standard and Sodium carbonate as inorganic carbon (IC) standard. The TOC content of the test substance was determined to be 54.48%.

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

46 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The IC50 for growth rate inhibition (96h-IRC50) was 62 mg/l (95% confidence interval between 23 and 170 mg/l), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l).
The IC50 for yield inhibition (96h-IYC50) was 40 mg/l (95% confidence interval between 19 and 83 mg/l), containing 30 mg a.i. per litre (95% confidence interval between 14 and 62 mg/l).
The IC50 for cell growth inhibition (96h-IBC50) was 39 mg/l (95% confidence interval between 22 and 68 mg/l), containing 29 mg a.i. per litre (95% confidence interval between 16 and 51 mg/l).
The 96h-NOEC for inhibition of growth rate, yield and cell growth was 13 mg/l, containing 9.7 mg a.i. per litre.
Incubation of subsamples taken from the highest test concentration in M2 medium without test substance showed that the inhibitory effect of Castor Oil, Sulfated, Sodium Salt, 75% was algistatic

Validity criteria fulfilled:

yes

Conclusions:

The IC50 for growth rate inhibition (96h-IRC50) was 62 mg/l (95% confidence interval between 23 and 170 mg/l), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l).

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with analogue substance 1.

The study procedure described in this report was based on the OCSPP 850.4500, 2012. In addition, the procedures were designed to meet the test methods of the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of analogue substance 1 tested was a clear amber liquid and completely soluble in test medium at the concentrations tested (or highly dispersible).

A final test was performed based on the results of a preceding combined limit/range-finding test. Preparation of test solutions started with a nominal concentration of 212 mg/l containing 158 mg of the active ingredient (a.i.) per litre. This concentration was magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 212 mg/l concentration in test medium. The final test solutions were clear and colourless.

Four replicates of exponentially growing algal cultures were exposed to a control and to nominal test substance concentrations of 1.4, 4.9, 17, 61 and 212 mg/l, containing 1.0, 3.7, 13, 46 and 158 mg a.i. per litre. The initial cell density was 104 cells/ml and the total exposure period was 96 hours. Samples for non-specific analytical confirmation of actual exposure concentrations were taken at the start, after 24 and after 96 hours of exposure. Subsequently, subsamples of the highest test concentration were incubated in M2 medium without test substance in order to determine if the effects on algal growth were algistatic or algicidal.

Non-specific analysis was used for the analytical support of the aquatic ecotoxicological studies, i.e. Total Organic Carbon (TOC) analysis was performed. Subsequently, the concentration of test substance in the aquatic samples was calculated using the TOC content of pure test substance (i.e. 54.48%). The initial concentrations in samples taken from nominal 1.4, 4.9, 17, 61 and 212 mg/l were calculated to be 0.30, 3.6, 15, 48 and 132 mg/l, i.e. 21-88% of nominal, respectively. The three highest test concentrations remained stable during the exposure period (100-113% of initial at the end of the test), while the lower test concentrations decreased to below the Limit Of Detection (LOD=0.01 mg/l) for the lowest test concentration and to 72% of initial for nominal 4.9 mg/l. Given these results, effect parameters were based on the average exposure concentrations: 0.089, 2.4, 13, 51 and 140 mg test substance per litre, containing 0.066, 1.8, 9.9, 38 and 104 mg a.i. per litre.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Analogue substance 1 inhibited growth rate, yield and cell growth of this fresh water algae species significantly at an average exposure concentrations 51 mg/l and higher. A test substance concentration of 51 mg/l contained 38 mg of the active ingredient per litre.

The IC50 for growth rate inhibition (96h-IRC50) was 62 mg/l (95% confidence interval between 23 and 170 mg/l), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l).

The IC50 for yield inhibition (96h-IYC50) was 40 mg/l (95% confidence interval between 19 and 83 mg/l), containing 30 mg a.i. per litre (95% confidence interval between 14 and 62 mg/l).

The IC50 for cell growth inhibition (96h-IBC50) was 39 mg/l (95% confidence interval between 22 and 68 mg/l), containing 29 mg a.i. per litre (95% confidence interval between 16 and 51 mg/l).

The 96h-NOEC for inhibition of growth rate, yield and cell growth was 13 mg/l, containing 9.7 mg a.i. per litre.

Incubation of subsamples taken from the highest test concentration in M2 medium without test substance showed that the inhibitory effect of analogue substance was algistatic.

Description of key information

IC50 = 46 mg/l (active ingredient)

Key value for chemical safety assessment

EC50 for freshwater algae:

46 mg/L

Additional information

Analogue substance 1 was slowly dissolved into water without breacking the unstable equilibrium that brings it into the water phase and a test on algae was performed:

The IC50 for growth rate inhibition (96h-IRC50) was 62 mg/l (95% confidence interval between 23 and 170 mg/l), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l).

Based on the read across considerations same results apply to target substance