Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 01 September 2015 and 20 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

yes

Remarks:

During the range-finding test, the temperature of the control media at 0 hours was measured to be slightly in excess of the 18 to 22 °C given in the study plan. This was considered not to affect the results of the test

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

See OECD 202

Principles of method if other than guideline:

- Principle of test: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. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a 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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

EC Number:

806-731-9

Cas Number:

1428353-74-5

Molecular formula:

Substance is a UVCB.

IUPAC Name:

Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

Test material form:

other: liquid

Details on test material:

Identification: TS13001Physical state/Appearance:amber colored liquidExpiry Date: 30 January 2015Storage Conditions:room temperature in the darkIntended use/Application: Lubricating oil additive

Specific details on test material used for the study:

Physical state/Appearance: Extremely viscous amber liquidBatch:Not providedPurity:100% productExpiry Date:12 March 2017Storage Conditions: Room temperature in the dark, under nitrogen.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Definitive Test: - Concentrations: loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/LSamples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates (replicates R1 – R4) at 48 hours for quantitative analysis. 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:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)Test Water:Reconstituted water (ISO medium) used for both the range-finding and definitive tests.Procedure:Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.Validation of Mixing PeriodPreliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. Increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.Range-finding test:The loading rates to be used in the definitive test were determined by a preliminary range-finding test.In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. 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. Visual observations made on the 100 mg/L loading rate WAF indicated that a significant amount of dispersed test item was present in the water column 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 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 to 23°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 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control group was maintained under identical conditions but not exposed to the test item. A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Definitive Test:Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.Experimental Preparation:Nominal amounts of test item (20, 36, 32, 56 and 100 mg) were each separately added to the surface of 20, 20, 10, 10, and 10 liters of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. 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 mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column 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 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions present.The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

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 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 by parthenogenesis. 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Nominal and measured concentrations:

Definitive test:Nominal: loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

Details on test conditions:

TEST SYSTEMExposure Conditions:As in the range-finding test 150 mL glass beakers containing approximately 100 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 to 22°C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. 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. TEST MEDIUM / WATER PARAMETERSThe reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.EFFECT PARAMETERS MEASUREDTest Organism Observations:Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.Water Quality Criteria:Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.The light intensity during the light period was measured using an ATP Instrumentation Lux meter.Vortex Depth Measurements:The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

3.2 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.No immobilization was observed at 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF.Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive test.Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations of 1.7, 7.2 and 61 mg/L. Measured concentrations of the 1.0, 10 and 100 mg/L test preparations at 48 hours had declined to 1.0, 4.7 and 53mg/L, indicating some instability of the test item.The temperature of the control at 0 hours was measured to be slightly in excess of the 18 to 22 °C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures did not deviate more than ± 1 °C throughout the test. DEFINITIVE TEST:Chemical Analysis of Test Loading Rates:Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.91 to 7.9 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.66 to 7.6 mg/L.The dissolved test item may have been one or several components of the test item. 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 nominal loading rates only.Immobilization Data:Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the following results:24 h EL50: 4.3 mg/L Loading Rate WAF (95% Confidence Limits: 3.7 - 5.1 mg/L Loading Rate WAF)48 h EL50: 4.0 mg/L Loading Rate WAF (95% Confidence Limits: 3.4 - 4.6 mg/L Loading Rate WAF)The No Observed Effect Loading rate after 24 and 48 hours exposure was 3.2 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 5.6 mg/L loading rate WAF. Sub-Lethal Effects:Some sub lethal effects were shown at 24 hour observations; five out of twenty daphnia in the control were observed to be trapped at the surface, however, at the 48 hour observations no trapping of the daphnia was observed. This was considered not to have adversely affected the results of the test.Validation Criteria:The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.Water Quality CriteriaTemperature was maintained at 21 to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.Throughout the test the light intensity was observed to be in the range 361 to 595 lux.Vortex Depth Measurements:The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.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 the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate were observed to be clear colorless water columns with lumps and strings of test item dispersed throughout the water column. After 23 hours stirring and a 1-Hour standing period the 1.0 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface and suspended throughout. The 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to remain clear colorless water columns with lumps and strings of test item dispersed throughout. Visual inspection of the WAFs showed test item dispersed throughout the water column 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 no micro dispersions of test item observed in any loading rate.At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

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 immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:24 hr EC50: 1.1 mg/L 24 hr NOEC: 0.56 mg/L24 hr LOEC: 1.0 mg/L48 hr EC50: 0.75 mg/L (95% confidence limits: 0.69 - 0.82 mg/L)48 hr NOEC: 0.56 mg/L48 hr LOEC: 1.0 mg/LThe No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Table 1     Cumulative Immobilization Data and Observations in the in the Range-finding Test

Nominal Loading Rate (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	2 N 8 T	0	3 N 7 T
1.0	0	10 N	0	10 N
10	10	A/I	10	A/I
100	10	A/I	10	A/I

N = No sub-lethal effects observed

T = Trapped at surface

A/I = All daphnia immobilized

Table 2     Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	2T 3N	3T 2N	5N
1.0	0	0	0	0	0	0	5N	5N	5N	5N
1.8	0	0	0	0	0	0	5N	5N	5N	5N
3.2	2	0	2	1	5	25	3N	5N	3N	4N
5.6	3	3	4	4	14	70	2N	2N	1N	1N
10	5	5	5	5	20	100	A/I	A/I	A/I	A/I

Nominal Loading Rate (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
1.0	0	0	0	0	0	0	5N	5N	5N	5N
1.8	0	0	0	1	1	5	5N	5N	5N	4N
3.2	2	2	0	0	4	20	3N	3N	5N	5N
5.6	4	5	4	4	17	85	1N	A/I	1N	1N
10	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

T = Trapped at surface

A/I = All daphnia immobilized

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:48 h EL50 (mg/L Loading Rate WAF): 4.0 48 h No Observed Effect Loading Rate (NOEL) (mg/L): 3.248 h Lowest Observed Effect Loading Rate (LOEL) (mg/l): 5.6

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