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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 02 September 2015 and 21 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

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:

Identification:[redacted]Description:Extremely viscous amber liquidBatch:TS13001Purity:100% productExpiry Date:12 March 2017Storage Conditions: Stored under nitrogen at room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Definitive Test: - Concentrations: loading rate of 2.6, 6.4, 16, 40, 100 mg/LWater samples were taken from the control and all surviving test groups at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) 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:The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.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 Period:Preliminary 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 fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 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. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations determined 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) and qualitative filter paper. 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. Microscopic observations were performed on the WAFs after filtering and showed no microdispersions in the 1.0 and 10 mg/L loading rates; however the 100 mg/L loading rate remained a cloudy homogenous dispersion.In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at approximately 14°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions. Each 25-30 liter test and control vessel contained 20 liters of test media and was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.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 24 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.Definitive Test:Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 2.6, 6.4, 16, 40 and 100 mg/L.Experimental Preparation:Prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (57.2, 140.8, 352, 880 and 2200 mg) were each separately added to the surface of 22 liters of test water to give the 2.6, 6.4, 16, 40 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. 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) and qualitative filter paper. A length of Tygon tubing was attached to the top of the glass siphon tube. 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 2.6, 6.4, 16, 40 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed fine particles in the 6.4, 16, 40 and 100 mg/L loading rates; however no particles were observed in the 2.6 mg/L loading rate.The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24, 72 and 96 hours.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout. Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 04 August 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 22 to 29 September 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 8.8 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.0 cm (sd = 0.87) and a mean weight of 1.21 g (sd = 0.57) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.42 g bodyweight/liter.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Nominal and measured concentrations:

Definitive test:Nominal: loading rate of 2.6, 6.4, 16, 40 and 100 mg/L

Details on test conditions:

TEST SYSTEMExposure conditions:25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.The control group was maintained under identical conditions but not exposed to the test item. A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the buildup of nitrogenous waste products.TEST MEDIUM / WATER PARAMETERSThe test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. EFFECT PARAMETERS MEASURED Test Organism Observations:Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.Water Quality Criteria:The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. 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. Vortex depth measurements:The vortex depth was recorded at the start and end of each mixing period.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

10.2 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

6.4 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

RANGE-FINDING TEST:Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 and sub-lethal effects of exposure are given in Table 2. The results showed no mortalities at 1.0 and 10 mg/L loading rate WAF, however, mortalities were observed at 100 mg/L loading rate WAF.After approximately 1.5 hours exposure all fish at 100 mg/L loading rate WAF were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) these fish were killed and classed as mortalities for the following observational time point.As the test item was observed to form a dispersion in the test diluent, microscopic inspection of the gill filaments of the dead or killed fish was performed. This examination revealed that no particles of test item had adhered to the gill filaments indicating physical toxicity was not a factor.Based on this information loading rates of 2.6, 6.4, 16, 40 and 100 mg/L were selected for the definitive test.Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations of 0.63 to 92 mg/L. Analysis of the old media at 24 hours showed measured test concentrations of 0.40 to 106 mg/L indicating some instability of the test item.DEFINITIVE TEST:Chemical Analysis of Test Loading Rates:Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations of 1.8 to 50 mg/L. Chemical analysis of the aged test preparations at 24 and 96 hours showed measured test concentrations to range from 1.7 to 49 mg/L.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 mixture of components but to the test item as a whole, the results were based on nominal loading rates only.Mortality Data:Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 3. Analysis of the mortality data by Probit analysis using Linear Maximum-Likelihood regression at 48 and 96 hours based on the nominal test concentrations gave the following results:Time (h)LL50 (mg/L Loading Rate WAF)18434462524174810.27210.29610.2The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 6.4 mg/L, the lowest loading rate resulting in 100% mortality to be 16 mg/L. The Lowest Observed Effect Loading rate (LOEL) was considered to be 16 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 6.4 mg/L loading rate WAF. Sub-Lethal Effects:Sub-lethal effects of exposure were observed at 2.6, 6.4, 16, 40 and 100 mg/L loading rate WAF. These responses were exaggerated respiration, swimming at the bottom, loss of equilibrium, increased pigmentation, lethargy and moribund (see Table 4).After approximately 1 hour exposure 6 out of 7 fish at 100 mg/L loading rate WAF were observed to exhibit severe sub-lethal effects. After approximately 3 hours the remaining fish at 100 mg/L loading rate and 2 out of 7 fish in the 40 mg/L loading rate were observed to be moribund. At approximately 6 hours of exposure the remaining fish in the 40 mg/L loading rate were observed to be moribund. At approximately 20 hours of exposure 1 out of 7 fish in the 16 mg/L loading rate was observed to be moribund and after approximately 24 hours exposure 2 out of 6 fish in the 16 mg/L loading rate were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) these fish were killed and classed as mortalities for the following observational time point.Observations on Test Animals:As the test item was observed to form a dispersion in the test diluent, microscopic inspection of the gill filaments of the dead or killed fish was performed. This examination revealed that no particles of test item had adhered to the gill filaments indicating physical toxicity was not a factor.Validation Criteria:The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was greater than or equal to 60% of ASV (6.1 mg O2/L) in the control and test vessels.Water Quality Criteria:Temperature was maintained at 13 to 15°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.Vortex Depth Measurements:The vortex depth was recorded at the start and end of each 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 2.6, 6.4, 16, 40 and 100 mg/L loading rates were observed to be clear colorless water columns with strands of test item on the bottom of the vessel. After 23 hours stirring and a 1-Hour standing period the 2.6, 6.4, 16, 40 and 100 mg/L loading rates were observed with fine particles of test item suspended in the water column with turbidity increasing with increasing concentration. Due to the visual observations it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length) and qualitative filter paper. Microscopic inspection of the WAFs after filtration showed no micro-dispersions or undissolved test item to be present in the 2.6 mg/L loading rate; however fine particles were observed in the 6.4, 16, 40 and 100 mg/L loading rates. During the test the control, 2.6, 6.4 and 16 mg/L loading rates were observed to be clear, colorless solutions; the 40 and 100 mg/L loading rates were observed to be cloudy homogenous dispersions with a foam layer on the surface.

Results with reference substance (positive control):

No positive control

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1     Cumulative Mortality Data in the Range-finding Test

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 3)
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0
100	0	3*	3	3	3	3	3

*3 fish observed as moribund at approximately 1.5 hours and humanely killed at this time, considered as mortalities at 3 hours.

Table  2     Sub-lethal Effects of Exposure in the Range-finding Test

Nominal Loading Rate (mg/L)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	3	3	3	3	3	3	3
1.0	No abnormalities detected	3	3	3	3	3	3	3
10	No abnormalities detected	3	3	3	3	0	0	0
	Increased pigmentation and sat at the bottom.	0	0	0	0	3	3	3
100	Increased pigmentation and at bottom of tank	3	0	A/D	A/D	A/D	A/D	A/D
	Moribund	0	3*	A/D	A/D	A/D	A/D	A/D

*          3 fish observed as moribund at approximately 1.5 hours and humanely killed at this time, considered as mortalities at 3 hours.

A/D    All fish dead

Table 3     Cumulative Mortality Data in the DefinitiveTest

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)							% Mortality
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
2.6	0	0	0	0	0	0	0	0
6.4	0	0	0	0	0	0	0	0
16	0	0	0	3^#	7	7	7	100
40	0	2^†	7^‡	7	7	7	7	100
100	6*	7^**	7	7	7	7	7	100

*          6 fish observed with loss of equilibrium and increased pigmentation at approximately 1 hour and humanely killed at this time, considered as mortalities at 1 hour.

^**              1 fish observed as moribund at approximately 3 hours and humanely killed at this time, considered as mortality at 3 hours.

^†          2 fish observed with loss of equilibrium at approximately 3 hours and humanely killed at this time, considered as mortalities at 3 hours.

^‡          5 fish observed as moribund at approximately 6 hours and humanely killed at this time, considered as mortalities at 6 hours.

^#          1 fish observed as moribund at approximately 20 hours and 2 fish observed as moribund at approximately
24 hours and humanely killed at these times, considered as mortalities at 24 hours.

Table 4     Sub-lethal Effects of Exposure in the DefinitiveTest

Nominal Loading Rate (mg/L)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7	7
2.6	No abnormalities detected	7	4	7	7	7	7	7
	Sitting at the bottom	0	3	0	0	0	0	0
6.4	No abnormalities detected	7	7	7	7	7	7	2
	Swimming at bottom	0	0	0	0	0	0	5
16	No abnormalities detected	7	7	7	0	A/D	A/D	A/D
	Swimming at the bottom	0	0	0	4
	Moribund	0	0	0	3^#
40	No abnormalities detected	0	1	0	A/D	A/D	A/D	A/D
	Exaggerated respiration	7	0	0
	Sitting / Swimming at bottom	4	4	0
	Moribund	0	2^†	5^‡
100	Loss of equilibrium and increased pigmentation	6*	0	A/D	A/D	A/D	A/D	A/D
	Lethargic	1	0
	Moribund	0	1^**

*          6 fish observed with loss of equilibrium and increased pigmentation at approximately 1 hour and humanely killed at this time, considered as mortalities at 1 hour.

^**              1 fish observed as moribund at approximately 3 hours and humanely killed at this time, considered as mortality at 3 hours.

^†          2 fish observed with loss of equilibrium at approximately 3 hours and humanely killed at this time, considered as mortalities at 3 hours.

^‡          5 fish observed as moribund at approximately 6 hours and humanely killed at this time, considered as mortalities at 6 hours.

^#          1 fish observed as moribund at approximately 20 hours and 2 fish observed as moribund at approximately
24 hours and humanely killed at these times, considered as mortalities at 24 hours.

A/D    All fish dead

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of the freshwater fish rainbow trout to the test item has been investigated and gave the following results:96 h LL50 (mg/L Loading Rate WAF): 10.296 h No Observed Effect Loading Rate (NOEL) (mg/L): 6.496 h Lowest Observed Effect Loading Rate (LOEL) (mg/l): 16