Phosphoric acid, decyl octyl ester

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Study period:

From September 11, 2011 to September 30, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

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

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test substance, 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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test substance in cases where the test substance 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 substance with water for a prolonged period. Pre-study work showed that a preparation period of 24 h was sufficient to ensure equilibration between the test substance and water phase. At the completion of mixing and following a 1 h standing period, the test substance 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 substance in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test substance in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations
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.

Sampling method
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 h (old media) for quantitative analysis. The 0 and 24 h samples were stored at approximately -20°C prior to analysis. Duplicate samples and samples at 24 (fresh media), 48 and 72 h (fresh and old media) were taken and stored at approximately -20°C for further analysis, if necessary.

Sample storage conditions before analysis
All samples were stored at approximately -20°C prior to analysis.

Vehicle:

no

Details on test solutions:

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. Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaC03. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (see attached background material).

Validation of mixing period
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances. A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 h and the other for a period of 95 h. After a 1 h standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see attached background material Appendix 3: Validation of Mixing Period).

Range finding test
Due to the low aqueous solubility and complex nature of the test substance for the purposes of the range-finding test the test substance was prepared as a Water Accommodated Fraction (WAF). 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 10 and 100 mg/lL. Amounts of test substance (210 and 2100 mg) were separately added to the surface of 21 L of dechlorinated tap water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test substance, 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 h and the mixtures allowed to stand for 1 h. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test substance 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 approximate 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro particles of test substance to be present.In the range-finding test three fish were added to each 20 L test and control vessel and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 minute dawn and dusk transition periods for a period of 96 h under static test conditions. The control group was maintained under identical conditions but not exposed to the test substance. Data from the control group was shared with similar concurrent studies. Each vessel was covered to reduce evaporation. After 3, 24, 48, 72 and 96 h any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. A sample of each loading rate WAF was taken for chemical analysis at 0 and 24 h in order to determine the stability of the test substance under test conditions. All samples were stored at approximately -20°C prior to analysis. The loading rate to be used in the definitive test only were analysed.

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.

Experimenral preparation
An amount of test substance (2100 mg) was added to the surface of 21 L of dechlorinated tap water to give the 100 mg/L loading rate. After the addition of the test substance, 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 h and the mixture allowed to stand for 1 h. Microscopic observations made on the WAF indicated that a significant amount of dispersed test substance 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 approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no micro particles of test substance to be present. This method of preparation was conducted in duplicate to give replicates R1 and R2. The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 h (old media).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 21 July 2011. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 14 September 2011 to 26 September 2011. The lighting cycle was controlled to give a 16 h light and 8 h 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 7.8 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 h prior to the start of the definitive test. There was zero mortality in the 7 d prior to the start of the test and the fish had a mean standard length of 5.7 cm (sd = 0.4) and a mean weight of 2.62 g (sd = 0.46) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.92 g bodyweight/litre. The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Test water with a total hardness of approximately 140 mg/L as CaC03.

Test temperature:

The test vessels were sealed and maintained at approximately 14ºC.

pH:

pH was recorded daily throughout the test. The pH of test solutions ranged from 7.1 to 8.1 from measurements taken at 0, 24, 48, 72 and 96 hours with fresh and old media.

Dissolved oxygen:

The dissolved oxygen concentration was measured using a dissolved oxygen meter.

Salinity:

Freshwater used.

Nominal and measured concentrations:

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.

Details on test conditions:

Exposure conditions
As in the range-finding test 20 L glass exposure vessels were used for each 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 14ºC in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 minute dawn and dusk transition periods for a period of 96 h. 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 substance. Data from the control group was shared with similar concurrent studies. A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure test concentrations of soluble components of the test substance were maintained over the test and to prevent the build up of nitrogenous waste products.

Effect parameter measured
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 h after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Physico-chemical measurements:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 h, and after each test media renewal at 24, 48 and 72 h, 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 h, represent those of the used or 24 h old test preparations. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter and the temperature was measured using Hanna Instruments HI 93510 digital thermometer.

Vortex depth measurement:
The vortex depth was recorded at the start and end of each mixing period.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

ca. 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Validation of mixing period
Pre-study investigational work (see attached background material Appendix 3: Validation of Mixing Period) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 h. Therefore, for the purpose of testing the test substance was prepared using a stirring period of 23 h followed by a 1 h settlement period.

Range finding test:
Cumulative mortality data from the exposure of rainbow trout to the test substance during the range-finding test are given in Table 1 (see Any other information on results incl. tables section). There were no sub-lethal effects of exposure during the range-finding test. The results showed no mortalities at 10 and 100 mg/L loading rate WAF. Based on this information, a single loading rate, in duplicate, of 100 mg/L using a stirring period of 23 h followed by a 1 h standing period was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no mortalities or sub-lethal effects of exposure were observed. Chemical analysis of the 100 mg/L loading rate WAF (see attached background material Appendix 4: Chemical analysis of test loading rates) showed a measured concentration of 10.3 mg/L at 0 h and 8.93 mg/L at 24 h indicating that the test substance was stable under test conditions.

Definitive test:
Mortality data: Cumulative mortality data from the exposure of rainbow trout to the test substance during the definitive test are given in Table 2 (see Any other information on results incl. tables section). There were no mortalities in 14 fish exposed to a 100 mg/L loading rate WAF for a period of 96 h. Inspection of the mortality data gave the following results: Time (h) LL50 (mg/l) 3 > 1006 > 10024 > 10048 > 10072 > 10096 > 100. The results of the definitive test showed the No Observed Effect Loading rate (NOEL) to be 100 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate. 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 in 14 fish exposed to a 100 mg/L loading rate WAF for a period of 96 h.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 5 (see any other information on results section). Temperature was maintained at approximately 14ºC throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed for the fresh media throughout the test.

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 (see Table 3 (any other information on results section).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs. At the start and end of each mixing period, and after the 1 h settlement period the 100 mg/L loading rate was observed to be a colourless water column with test substance dispersed throughout. Microscopic examination of the WAF showed microscopic particles of test substance to be present and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed no microscopic particles of test substance to be present. At 0 h the 100 mg/L loading rate WAF was observed to be a very slightly cloudy homogenous dispersion. For the freshly prepared 24, 48 and 72 h the 100 mg/L loading rate WAF was observed to be a clear, colourless solution however, the 24 h old media at 24, 48, 72 and 96 h were observed to be a cloudy, white homogenous dispersions.

Chemical analysis of test loading rates
Analysis of the 100 mg/L loading rate WAF at 0 (fresh media), 24 and 96 h (old media) (see attached background material Appendix 4: Chemical analysis of test loading rates) showed measured concentrations to range from 8.52 to 13.1 mg/L. The dissolved test substance may have been one or several components of the test substance. Given that toxicity cannot be attributed to a single component or mixture of components but to the test substance as a whole, the results were based on nominal loading rates only.

Validation Criteria
In the control, no mortalities were observed over the 96 h test period. Thus the validity criterion was met. The oxygen content was ≥ 85% Air Saturated Value in the control throughout the test. Thus the validity criterion was met.

Results with reference substance (positive control):

No positve control used in study.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality data in the range-finding test

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 3)
	3 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0
100	0	0	0	0	0
100	0	0	0	0	0

 

Table 2: Cumulative mortality data in the definitive test

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 h	6 h	24 h	48 h	72 h	96 h	96 h
Control	0	0	0	0	0	0	0
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

 

Table 3: Vortex depth measurements at the start and end of each mixing period - First mixing period

	Nominal Loading Rate (mg/L)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	34.5	34.5	34.5	34.5	34.5	34.5
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

Second mixing period

	Nominal Loading Rate (mg/L)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	34.5	34.5	35.0	35.0	35.0	35.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

Third mixing period

	Nominal Loading Rate (mg/L)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	34.5	34.5	35.0	35.0	35.0	35.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

Fourth mixing period

	Nominal Loading Rate (mg/L)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	34.5	34.5	35.0	35.0	35.0	35.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

R₁- R₂= Replicates 1 and 2

*= Start of mixing period

+= End of mixing period

Appendix 5: Physico-chemical measurements

Nominal Loading Rate (mg/L)	Time (h)
	0 h (fresh media)				24 h (old media)				24 h (fresh media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.0	9.6	95	15	8.3	10.1	98	14	8.0	9.4	93	15
100 R1	7.2	9.7	96	15	8.0	8.9	86	14	7.2	9.5	94	15
100 R2	7.1	9.4	93	15	8.0	9.3	90	14	7.1	9.6	95	15

 

Nominal Loading Rate (mg/L)	Time (h)
	48 h (old media)				48 h (fresh media)				72 h (old media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.0	9.9	96	14	7.9	8.8	85	14	8.5	9.9	96	14
100 R1	8.1	9.6	93	14	7.1	9.5	94	15	8.1	9.4	93	15
100 R2	8.0	9.3	90	14	7.1	9.7	96	15	8.1	9.0	87	14

 

Nominal Loading Rate (mg/L)	Time (h)
	72 h (fresh media)				96 h (old media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C
Control	7.8	9.9	98	15	8.4	9.8	95	14
100 R1	7.2	9.7	94	14	8.2	9.6	93	14
100 R2	7.1	9.8	95	14	8.1	9.5	92	14

R₁

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of read across study, a 96 h LL50 and NOELR values for the test substance, mono- and di- C8-10 PSE can be considered to be >100 mg/L and 100 mg/L (nominal loading rate WAF), respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, mono- and di- C6-10 PSE (Purity not specified) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C1, in compliance with GLP. Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the substance, at a single nominal loading rate of 100 mg/L for a period of 96 h at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 h after the start of exposure and then daily throughout the test until termination after 96 h. Analysis of the 100 mg/L loading rate WAF at 0 (fresh media), 24 and 96 h (old media) showed measured concentrations to range from 8.52 to 13.1 mg/L.The dissolved read across substance may have been one or several components of the read across substance. Given that toxicity cannot be attributed to a single component or mixture of components but to the read across substance as a whole, the results were based on nominal loading rates only.The 96 h LL50 (lethal loading rate) based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate (NOELR) was 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.Under the study conditions, the 96 h LL50 and NOELR values for the read across substance were determined to be >100 mg/L and 100 mg/L (nominal), respectively (XXXX, 2012). Based on the results of read across study, similar 96 h LL50 and NOELR values can be expected for the test substance, mono- and di- C8-10 PSE.

Description of key information

Based on the results of read across study, the 96 h LL50 and NOELR values for the test substance, mono- and di- C8-10 PSE can be considered to be >100 mg/L and 100 mg/L (nominal loading rate WAF), respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

A study was conducted to determine the acute toxicity of the read across substance, mono- and di- C6-10 PSE (Purity not specified) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C1, in compliance with GLP. Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the substance, at a single nominal loading rate of 100 mg/L for a period of 96 h at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 h after the start of exposure and then daily throughout the test until termination after 96 h. Analysis of the 100 mg/L loading rate WAF at 0 (fresh media), 24 and 96 h (old media) showed measured concentrations to range from 8.52 to 13.1 mg/L. The dissolved read across substance may have been one or several components of the read across substance. Given that toxicity cannot be attributed to a single component or mixture of components but to the read across substance as a whole, the results were based on nominal loading rates only. The 96 h LL50 (lethal loading rate) based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate (NOELR) was 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.Under the study conditions, the 96 h LL50 and NOELR values for the read across substance were determined to be >100 mg/L and 100 mg/L (nominal), respectively (XXXX, 2012). Based on the results of read across study, similar 96 h LL50 and NOELR values can be expected for the test substance, mono- and di- C8-10 PSE.