Paraffin waxes and Hydrocarbon waxes, oxidized, lithium salts

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24th April 2012 to 14th June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom.

Analytical monitoring:

yes

Details on sampling:

Samples of the control and 100 mg/l loading rate WAF (Water Accommodated Fraction) test group for the definitive test and the control, 10 and 100 mg/l loading rate WAF test groups for the additional experiment were taken for analysis at 0 (fresh media) and 48 hours (old media).

-Samples were stored at approximately -20 °C prior to analysis.
-Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a WAF of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

RANGE-FINDING TEST
-Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a WAF.
-Amounts of test material (10, 40 and 250 mg) were each separately added onto glass slides and suspended in the water column of 10, 4 and 2.5 litres of reconstituted water to give the 1.0, 10 and 100 mg/l loading rates respectively. After the addition of the test item, the reconstituted 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 mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column of the 10 mg/l loading rate WAF and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 - 4 cm in length).
-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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 - 100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-particles to be present.

DEFINITIVE TEST
-Amounts of test item (10, 18, 32, 56, 40, 90, 80, 140 and 250 mg) were each separately added onto glass slides and suspended in the water column of 10, 10, 10, 10, 5, 5, 2.5, 2.5 and 2.5 litres of reconstituted water to give the 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l loading rates respectively.
-After the addition of the test item, the reconstituted 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 mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column of the 10 to 100 mg/l loading rate WAFs and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 - 4 cm in length).
-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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 - 100 ml discarded) to give the 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microscopic particles to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Source: Derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): 1st instar Daphnia. 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.
- Method of breeding: Parthenogenesis.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium 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.
- Type and amount of food: Each culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Feeding frequency: Daily.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

The reconstituted water in which the test was conducted had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

21 ± 1 °C

pH:

Measured daily throughout the test and found to be in the range 7.9 - 8.0.

Dissolved oxygen:

Measured daily throughout the test and found to be in the range 8.4 - 8.8 mgO2/l.

Salinity:

NDA

Nominal and measured concentrations:

Range-finding Test
-Nominal concentrations of 1.0, 10 and 100 mg/l.

Definitive Test
-Nominal concentrations of 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l.

Additional Test
-Nominal concentrations of 10 and 100 mg/l.

Details on test conditions:

RANGE-FINDING TEST
-Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a WAF.
- 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 °C to 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 250 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
-The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
-Loading rates were assigned based on the range-finding test.
-Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 48 hours. Only the test preparations at the No Observed Effect Loading Rate (NOEL) were analysed.

Exposure Conditions
-In the definitive test, 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 °C to 22 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity of 789 to 800 lux.
-The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
-The control group was maintained under identical conditions but not exposed to the test item.
-The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Vortex Depth Measurements
-The vortex depth was recorded at the start and end of the mixing period.

ADDITIONAL EXPERIMENT
-The results from the range-finding and definitive tests differed. An additional experiment was therefore conducted.

-An amount of test material (25 mg) was added onto a glass slide and suspended in the water column of 2.5 litres of reconstituted water to give the 10 and 100 mg/l loading rates. A further amount of test item (250 mg) was added to the surface of 2.5 litres of reconstituted water to give the 100 mg/l loading rate.
-After the addition of the test item, the reconstituted 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 mixtures 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 WAFs 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 10 and 100 mg/l loading rate WAFs.

- 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
-The control group was maintained under identical conditions but not exposed to the test item.
-Samples of the control, 10 and 100 mg/l loading rate WAF test groups were taken for TOC analysis at 0 and 48 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
-Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.
-No immobilisation was observed at 1.0 mg/l loading rate WAF. However, immobilisation was observed at 10 and 100 mg/l loading rate WAF.

DEFINITIVE TEST
-Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.
-There was no immobilization throughout the duration of the test.
-It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

OBSERVATIONS ON TEST ITEM SOLUBILITY
-Observations on the test media were carried out during the mixing and testing of the WAFs.
-At the start of the mixing period all the loading rates were observed to be clear, colourless water columns with brown test item glass slides suspended in the water columns. After 23 hours stirring and a 1 hour standing period the 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l loading rates were observed to remain clear, colourless water columns with brown test item glass slides suspended in the water columns.
-Microscopic examination of the WAFs showed particles to be visible in the 10 to 100 mg/l loading rate WAFs and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 - 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all the microscopic particles. During the test the 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l loading rates were observed to be clear, colourless solutions.

TOTAL ORGANIC CARBON ANALYSIS
-Total Organic Carbon (TOC) analysis of the 100 mg/l loading rate WAF at 0 and 48 hours showed concentrations of 3.36 and 3.26 mg carbon/l respectively.
-The dissolved test item may have been one or several components of the test item. Given that 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 nominal loading rates only.

ADDITIONAL EXPERIMENT
-Cumulative immobilization data from the exposure of Daphnia magna to the test item during the additional experiment are given in Table 3.
-No immobilization was observed at 10 and 100 mg/l loading rate WAF.
-Total Organic Carbon (TOC) analysis of the 10 and 100 mg/l loading rate WAF at 0 and 48 hours showed concentrations of less than the limit of quantitation (assessed as 1.0 mg carbon/l) to 2.15 mg carbon/l respectively.
-The results from the additional experiment were comparable to those obtained during the definitive test. It was therefore considered that the immobilisation observed during the range-finding test was not due to toxicity attributable to the test item.

Results with reference substance (positive control):

-A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
-Exposure conditions for the positive control were similar to those in the definitive test.

-Analysis of the immobilisation data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95 % Confidence limits (mg/l)
24 1.3 1.0 - 1.8
48 1.1 1.0 - 1.3

-The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.56 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

-The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table 1 Cumulative Immobilisation Data in the Range-finding Test

Nominal loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	5
100	4	8

 

Table 2 Cumulative Immobilisation Data in the Definitive Test

Nominal loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 5 per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0	0	0	0	0
5.6	0	0	0	0	0	0	0	0	0	0	0	0
8.0	0	0	0	0	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0	0	0	0	0
56	0	0	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0	0	0

R1 - R4 = Replicates 1 to 4

 

Table 3 Cumulative Immobilisation Data in the Additional Experiment

Nominal loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 per Replicate)
	24 Hours	48 Hours
Control	0	0
10	0	0
100	0	0

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to Daphnia magna has been investigated and gave a 48-Hour EL50 value based on nominal loading rates of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

In a GLP compliant study performed according to the standardised guidelines OECD 202 and Method C.2 of Commission Regulation (EC) No. 440/2008, the toxicity of the test material was determined in a Daphnia Acute Toxicity Test.

Following a preliminary range-finding test, twenty daphnids were exposed to Water Accommodated Fractions (WAFs) of the test material, over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 8.0, 18, 32, 56 and 100 mg/l for a period of 48 hours at a temperature of approximately 21 °C under static test conditions.

The 48-Hour EL50 based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Description of key information

The toxicity of the test material was determined in Daphnia magna. Under the conditions of the test, no toxic effects were noted and the 48-Hour EL50 based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The potential for hydrocarbon waxes to cause acute toxic effects to aquatic invertebrates was determined in a GLP compliant study (Goodband, 2012b) performed according to the standardised guidelines OECD 202 and Method C.2 of the Commission Regulation (EC) No. 440/2008. The study was reported in sufficient detail to provide an accurate assessment of the results and study quality.

The study was performed to a good standard and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997).