Reaction products of petrolatum (petroleum), oxidized, esterified with methanol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2012 to 7 June 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 the 100 mg/l loading rate WAF (Water Accommodated Fraction) test group were taken for analysis at 0 (fresh media), 24 and 96 hours (old media).

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 23 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour standing 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
An amount of test item (11 mg) was added to a glass slide and suspended in the water column of 11 litres of dechlorinated tap water to give the 1.0 mg/l loading rate. Further amounts of test item (110 and 1100 mg) were each separately added to the surface of 11 litres of dechlorinated tap water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the dechlorinated tap 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 WAF was removed by mid-depth siphoning (the first 75 - 100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs.

DEFINITIVE TEST
An amount of test item (2100 mg) was added onto a glass slide and suspended in the water column of 21 litres of dechlorinated tap water to give the 100 mg/l loading rate. After the addition of the test item, the dechlorinated tap 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. Visual observations made on the 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 WAF 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 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed that all of the microscopic particles of test item had been removed by the glass wool plug.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: Oncorhynchus mykiss
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK.
- Age at study initiation: Juvenile.
- Length at study termination (mean and SD): standard length 4.2 cm (SD = 0.2).
- Weight at study termination (mean and SD): 1.08 g (SD = 0.19).
- Food type: Commercial trout pellets.
- Feeding during test: No; feeding was discontinued 24 hours prior to the start of the test.

ACCLIMATION
- Acclimation period: 12 days.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Commercial trout pellets.
- Health during acclimation (any mortality observed): less than 1 % mortality in the 7 days preceding the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not observed

Hardness:

Approximately 140 mg/l as CaCO3.

Test temperature:

14 ± 1 °C

pH:

Measured daily throughout the test and found to be in the range 7.7 - 8.3.

Dissolved oxygen:

Measured daily throughout the test and found to be in the range 9.8 - 10.2 mgO2/l.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Salinity:

NDA

Nominal and measured concentrations:

A single concentration of 100 mg/L as a WAF.

Details on test conditions:

-The test solutions consisted of laboratory tap water that was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener).

RANGE-FINDING TEST
-The loading rate to be used in the definitive test was 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.
-In the range-finding test 3 fish were added to each 10 litre test and control vessel 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 under static test conditions.
-The control group was maintained under identical conditions but not exposed to the test item.
-Each vessel 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.

DEFINITIVE TEST
-Based on the results of the range-finding test a limit test was conducted at a single loading rate of 100 mg/l to confirm that no mortalities or sub-lethal effects of exposure were observed.
-Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 (fresh media), 24 and 96 hours (old media).

Exposure Conditions
-In the definitive test, 20 litre glass exposure vessels were used for each test concentration. At the start of the test, 7 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 ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
-Any mortalities and sub-lethal effects of exposure were recorded at 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.

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

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

RANGE-FINDING TEST
-There were no mortalities at 10 and 100 mg/l loading rate WAF.
-A single mortality was observed in the control and 1.0 mg/l loading rate WAF however, these mortalities were considered to be possibly due to injury rather than a toxic effect.
-Based on this information, a single loading rate of 100 mg/l using a stirring period of 23 hours followed by a 1-Hour standing period was selected for the definitive test.

DEFINITIVE TEST
Mortality Data
-There were no mortalities in 7 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours.
-The results showed the highest loading rate resulting in 0 % mortality to be greater than or equal to 100 mg/l loading rate WAF. The No Observed Effect Loading rate (NOEL) was 100 mg/l loading rate WAF.
-It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Sub-Lethal Effects
-There were no sub-lethal effects of exposure observed.

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 each mixing period, the 100 mg/l loading rate was observed to be a clear, colourless water column with test item on a glass slide suspended in the water column. At the end of each mixing period, and after the 1-Hour settlement period, the 100 mg/l loading rate was observed to be a clear, colourless water column with test item on a glass slide suspended in the water column along with particles of test item dispersed throughout. Visual inspection of the WAF showed particles of test item to be present. After siphoning through a glass wool plug, and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution.

Total organic carbon analysis
-Samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media) and 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method.
-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, and the dissolved test item was close to or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

NDA

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to Oncorhynchus mykiss has been investigated and gave a 96-Hour LL50 value based on nomonal 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 203 and Method C.1 of Commission Regulation (EC) No. 440/2008, the toxicity of the test material was determined in a Fish Acute Toxicity Test.

Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of approximately 14 °C under semi-static test conditions.

The 96-Hour LL50 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.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See read-across justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 June 1999 to 20 June 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

A Water Accomodated Fraction (WAF) of test material was prepared according to ASTM D6081-97 (modified).

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A Water Accomodated Fraction (WAF) of test material was prepared according to ASTM D6081-97 (modified).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Length at study initiation: 5.0 ± 1.0 cm
- Feeding during test: fish are not fed for 24 hours before, nor during the test

ACCLIMATION
- Acclimation period: 12 days minimum
- Acclimation conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

15.1 - 15.9 °C

pH:

7.26 - 8.23

Dissolved oxygen:

3.8 - 10.0

Nominal and measured concentrations:

0, 1, 10, 100, 1000, 10000 mg/L (preliminary study)
0, 625, 1250, 2500, 5000, 10000 mg/L (definitive study)

Details on test conditions:

TEST SYSTEM
- Test volume: 1800 mL (preliminary study), 4 L (definitive study)
- Aeration: yes, if DO < 60% (6.5 mL/min/L)
- No. of organisms per vessel: 3 (preliminary study), 7 (definitive study)
- No. of vessels per concentration (replicates): 1 replicate
- No. of vessels per dilution water control (replicates): 1 replicate
- Biomass loading rate: 0.95 g/L (definitive study)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 ± 1 hour light, 8 ± 1 hours darkness
- Light intensity: ~ 300 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): impairment and mortality were recorded daily

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 1, 10, 100, 1000, 10000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium chloride

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

3 540 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 2500 - 5000 mg/L (95% CL)

Results with reference substance (positive control):

- Reference substance data: The 96 hour LC50 value was determined to be 4070 mg/L (3590 - 4620 mg/L, 95 % CL) The results indicate that test reproducability and sensitivity of organisms were within established limits.

- Historic Mean LC50 of reference substance: 3800 mg/L (3280 - 4730 mg/L, 95 % CL)

Sublethal observations / clinical signs:

Table 1: Preliminary Study - Results

Time (hours)	Parameters	Control (shaken)	Test material loading (mg/L)
			Control	1	10	100	1000	10000
0	DO (mg/L)	9.2	10.1	10.1	10.0	10.1	10.1	8.8
	pH (initial)	7.85	8.07	8.13	8.15	8.16	8.14	8.02
	Temperature (°C)	14.1	14.7	14.7	14.7	14.7	17.9	15.8
	Cond. (µmhos/cm)	311	307	314	314	314	316	341
24	DO (mg/L)							8.6
	pH (initial)							7.65
	Temperature (°C)							15.3
	Cond. (µmhos/cm)							342
	Mortality	0	0	0	0	No fish	0	3
48	Mortality	0	0	0	0	No fish	0
72	Temperature (°C)	14.6	14.4	14.1	14.6		14.6
	Mortality	0	0	0	0	No fish	0
96	DO (mg/L)	6.2	6.4	6.0	6.0	9.9	6.0
	pH (initial)	7.26	7.36	7.36	7.33	8.00	7.32
	Temperature (°C)	15.7	15.6	15.6	15.6	15.9	15.8
	Cond. (µmhos/cm)	321	324	328	330	314	329
	Mortality	0	0	0	0	No fish	0

 

Table 2: Definitive Study - Results

Time (hours)	Parameters	Control (mix)	Test material loading (mg/L)
			Control	625	1250	2500	5000	10000
0	DO (mg/L)	9.6	9.4	9.9	9.9	10.0	9.4	8.6
	pH	7.89	7.73	7.97	7.99	8.00	8.07	8.23
	Cond. (µmhos/cm)	416	333	339	336	319	338	365
24	DO (mg/L)	6.9	6.9	6.9	7.2	7.0	6.2	3.8
	pH	7.53	758	7.50	7.60	7.60	7.55	7.35
	Cond. (µmhos/cm)	327	410	415	425	425	451	480
	Impairment	0	0	0	0	0	0	0
	Mortality	0	0	0	0	0	7	7
48	DO (mg/L)	8.2	8.0	7.6	8.0	7.5
	pH	7.45	7.26	7.33	7.48	7.38
	Cond. (µmhos/cm)	307	280	294	294	300
	Impairment	0	0	0	0	0
	Mortality	0	0	0	0	0
72	DO (mg/L)	8.2	7.9	7.0	8.0	7.2
	pH	7.64	7.69	7.51	7.66	7.53
	Cond. (µmhos/cm)	467	467	467	460	470
	Impairment	0	0	0	0	0
	Mortality	0	0	0	0	0
96	DO (mg/L)	8.6	8.4	7.5	8.4	7.4
	pH	7.56	7.55	7.45	7.62	7.54
	Cond. (µmhos/cm)	437	443	445	446	456
	Impairment	0	0	0	0	0
	Mortality	0	0	0	0	0

Sample was shaken at 110 rpm for 24 hours, then settled for 4 hours prior to testing 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the LL50 of the test material was determined to be 3540 mg/L with 95 % confidence limits of 2500 - 5000 mg/L. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The acute toxicity of the test material to fish was investigated in rainbow trout in accordance with the standardised guideline OECD 203. The fish were exposed to a Water Accommodated Fraction (WAF) of test material which was prepared according to a modified ASTM method. Following a preliminary study, groups of 7 fish were exposed to test material at the following concentrations: 0, 625, 1250, 2500, 5000 and 10000 mg/L. Potassium chloride was used as the reference substance.

In the definitive study, no mortalities were observed up to 2500 mg/L. All the fish died at the 5000 and 10000 mg/L dose levels. Under the conditions of the study, the LL50 of the test material was determined to be 3540 mg/L with 95 % confidence limits of 2500 - 5000 mg/L. The LC50 of the reference substance was determined to be 4070 mg/L with 95 % confidence limits of 3590 - 4620 mg/L. The reference substance results indicate that test reproducibility and sensitivity of the organisms were within established limits.

Description of key information

LL50 of structural analogue (EC 265-205-1) >100 mg/l loading rate WAF, rainbow trout (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Key Study

The potential for a structurally analogous substance (EC 265-205-1) to cause acute toxic effects to fish was determined in a GLP compliant study (Goodband, 2012a) performed according to the standardised guidelines OECD 203 and Method C.1 of the Commission Regulation (EC) No. 440/2008. The study was reported in sufficient detail so as to accurately represent the toxicity of the material.

The toxicity of the test material was determined in Oncorhynchus mykiss. Under the conditions of the test, no toxic effects were noted and the 96-Hour LL50 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.

The study was performed to a good standard and was assigned a reliability score of 2 using the principles for assessing data quality as set out in Klimisch (1997) as a result of the data being used in a read across capacity. Read across is justified on the basis of similar chemical structures and analogous results from a batch of physico-chemical tests, including water solubility.

Supporting Information

The acute toxicity of an analogue test material (EC 271 -238 -7) to fish was investigated in rainbow trout in accordance with the standardised guideline OECD 203. The fish were exposed to a Water Accommodated Fraction (WAF) of test material which was prepared according to a modified ASTM method. Following a preliminary study, groups of 7 fish were exposed to test material at the following concentrations: 0, 625, 1250, 2500, 5000 and 10000 mg/L. Potassium chloride was used as the reference substance.

In the definitive study, no mortalities were observed up to 2500 mg/L. All the fish died at the 5000 and 10000 mg/L dose levels. Under the conditions of the study, the LL50 of the test material was determined to be 3540 mg/L with 95 % confidence limits of 2500 - 5000 mg/L. The LC50 of the reference substance was determined to be 4070 mg/L with 95 % confidence limits of 3590 - 4620 mg/L. The reference substance results indicate that test reproducibility and sensitivity of the organisms were within established limits.

The non-GLP study was performed in line with an accepted standardised guideline with a sufficient level of reporting to assess the quality of the relevant results. The study was assigned a reliability score of 2 and considered suitable for assessment as an accurate reflection of the test material. However, it was deemed more appropriate to select the more reliable study conducted on a structural analogue as the key data to be used.