Alcohols, lanolin

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 2013 to 22 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

1) temperatures in the definitive test exceeded the maximum deviation of ±2⁰C; 2) coefficient of variation in the control group 27%

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Principles of method if other than guideline:

Both deviations were not considered to have had an effect on the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, lanolin

EC Number:

232-430-1

EC Name:

Alcohols, lanolin

Cas Number:

8027-33-6

Molecular formula:

UVCB

IUPAC Name:

Alcohols, lanolin

Test material form:

other: yellow solid

Details on test material:

- Name of test material (as cited in study report): Lanolin Alcohols
- Analytical purity: UVCB >90%
- Lot/batch No.: 0000664004
- Expiration date of the lot/batch: 19 February 2014
- Storage condition of test material:room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control the 100 mg/L loading rate (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 14, 16 and 19 and of the expired test preparations on Days 2, 7, 16, 19 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

An amount of test item (1000 mg) was added to the surface of 10 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis on days 0, 5, 14, 16 and 19 (fresh media) and days 2, 7, 16, 19 and 21 (old media)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 2) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin®flake food suspension. Culture conditions ensured that reproduction was byparthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Test conditions

Hardness:

240 to 260 mg/L CaCO3

Test temperature:

21(±1)°C

pH:

7.6-8.2

Dissolved oxygen:

> 8 mg/L O2

Nominal and measured concentrations:

Nominal: 0 and 100 mg/L

Details on test conditions:

For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL Glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at approximately 21 °C with a photoperiod of 16 hours light (514 to 709 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each vessel was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.

Each daphnid received approximately 2 to 7 µL of an algal suspension (Desmodesmus subspicatus) and approximately 10 to 32 µL of Tetramin®flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.

The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks. At the end of the test, the length of each surviving parent animal was determined.

Temperature of the test preparations and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each test concentration. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week. The temperature was measured in control hourly throughout the test using a Testo Temperature logger.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Duration:

21

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

reproduction

Details on results:

See tables below

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

The EL50 (immobilization) values up to Day 21 of the test were estimated by inspection of the data.
The EL50 (reproduction) value after 21 days was estimated by inspection of the data.
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) the numbers of live young produced per adult over the duration of the test for the control, and the 100 mg/L loading rate WAF test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) (see Appendix 5). Results from the control and the 100 mg/L loading rate WAF test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using 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) (see Appendix 5). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table: Analytical results for main test

Time point [hours]	Nominal Loading rate [mg/L]	Determined Concentration of Test Item in Test Sample[ mg/L]
Day 0 (Fresh)	Control 100	<LOQ 0.0385
Day 2 (Old)	Control 100	<LOQ 0.0174
Day 5 (Fresh)	Control 100	<LOQ 0.0289
Day 7 (Old)	Control 100	<LOQ <LOQ
Day 14 (Fresh)	Control 100	<LOQ 0.0291
Day 16 (Old)	Control 100	<LOQ <LOQ
Day 16 (Fresh)	Control 100	<LOQ 0.0540
Day 19 (Old)	Control 100	<LOQ 0.0137
Day 19 (Fresh)	Control 100	<LOQ 0.0176
Day 21 (Old)	Control 100	<LOQ <LOQ

Analysis of the 100 mg/L loading rate WAF on days 0, 5, 14, 16 and 19 (fresh media) showed measured concentrations to range from 0.018 to 0.054 mg/L. A decline in measured concentration was observed in the corresponding old media on days 2, 7, 16, 19 and 21 to between less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0098 mg/L and 0.017 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate only.

 

Table: Summary of findings following the exposure of Daphnia magna for 21 days

Nominal Loading Rate (mg/L)	% Survival of P1	Number of Live Young		Number of Dead Young		Number of Unhatched Egg
		Total	Per Female (cumulative)	Total	Per Female (cumulative)	Total	Per Female (cumulative)
Control	100	1408	141	0	0	0	0
100	100	1338	134	0	0	0	0

 

The "No Observed Effect Loading Rate" (NOEL) was 100 mg/L loading rate WAF as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes