Phosphoric acid, 2-ethylhexyl ester

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 7 June 2011 and 25 June 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions

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

Principles of method if other than guideline:

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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (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. 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.Validation of mixing period:Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis purified water. One loading rate was stirred for a period of 23 hours and the other for a period of 71 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the 100 mg/l loading rate WAF was verified by chemical analysis (see details on analyitcal methods section).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Single nominal loading rate of 100 mg/l.The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see details on analytical methods section).- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis. The method of analysis, recovery and test preparation analyses are described in details on analytical methods section.- Sample storage conditions before 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)- Method: Due to the low aqueous solubility and complex nature of the test item for the purposes of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).An 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 mixture 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.- Eluate: Not applicable- Controls: 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.- Evidence of undissolved material : Observations on the test media were carried out during the mixing and testing of the WAF.At the start of the mixing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with oily globules of test item dispersed throughout. At the end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with an oily layer of test item on the surface. Microscopic inspection of the WAF showed no microscopic particles of test item to be present. At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.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 (see Table 3 in any other information on results including tables section).

Test organisms (species):

Daphnia magna

Details on test organisms:

Test SpeciesThe 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21 to 22ºC throughout the test.Some of the temperatures were measured to be slightly in excess of the 20 ± 1°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 in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HClThe pH levels were measured using a Hach HQ30d Flexi Handheld meter.There was no treatment related differences for pH.The results of the physico-chemical measurements are given in any other information on results including tables section.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter.The results of the physico-chemical measurements are given in the any other information on results including tables section.

Salinity:

Freshwater used.

Nominal and measured concentrations:

Nominal: Single loading rate of 100 mg/l.Measured: Analysis of the test preparations at 0 hours (see details on analytical methods section) showed measured concentrations of of 96.8 mg/l and 97.2 mg/l in replicates R1 and R2 of the 100 mg/l loading rate WAF respectively. Measured concentrations of 96.4 mg/l and 98.2 mg/l were observed in replicates R1 and R2 respectively of the 100 mg/l loading rate WAF at 48 hours.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.

Details on test conditions:

TEST SYSTEMAs in the range-finding 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 to 22ºC with a photoperiod of 16 hours light 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. 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. - Aeration: None.- Source/preparation of dilution water:Reconstituted Water – Elendt M7 MediumSolution Concentration of stock solution (mg/l)(I)H3BO357190(II)MnCl2.4H2O7210(III)LiCl6120(IV)RbCl1420(V)SrCl2.6H2O3040(VI)NaBr320(VII)Na2MoO4.2H2O1260(VIII)CuCl2.2H2O335(IX)ZnCl2260(X)CoCl2.6H2O200(XI)Kl65(XII)Na2SeO343.8(XIII)NH4VO311.5(XIV)Na2EDTA.2H2O5000 FeSO4.7H2O1991An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21ºC.Macro Nutrient Stock SolutionsSolution Concentration of stock solution (g/l)(I)CaCl2.2H2O293.80(II)NaHCO364.80(III)MgSO4.7H2O246.60(IV)Na2SiO3.9H2O50.00(V)KCl58.00(VI)NaNO32.74(VII)K2HPO41.84(VIII)KH2PO41.43Vitamin NutrientsSolution Concentration of stock solution (mg/l)(IX)Thiamine hydrochloride750Cyanocobalamine (vitaminB12) 100D(+) biotin (vitamin H)75The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water. The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21ºC.Reconstituted Wateri)Stock Solutionsa)CaCl2.2H2O11.76 g/lb)MgSO4.7H2O4.93 g/lc)NaHCO32.59 g/ld)KCl0.23 g/lii)PreparationAn aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The 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.- Culture medium different from test medium:Reconstituted water was used for both the range-finding and definitive tests. The reconstituted water is defined in above.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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.TEST CONCENTRATIONS- Range finding study: Yes; The loading rate to be used in the definitive test was determined by a preliminary range-finding test. - Test concentrations: In the range-finding teest Daphnia magna were exposed to a series of nominal loading rates of 10 to 100 mg/l.. - Results used to determine the conditions for the definitive study: 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 immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/l LR WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l LR WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Details on results:

- Other adverse effects control: No other effects observed.- Abnormal responses: None recorded- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAF.At the start of the mixing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with oily globules of test item dispersed throughout. At the end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with an oily layer of test item on the surface. Microscopic inspection of the WAF showed no microscopic particles of test item to be present. At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless 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 immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the followingresults:Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)24 1.5 1.3 - 1.848 0.99 0.85 - 1.1The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. 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.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table1 (below).

No immobilisation was observed at 10 and 100 mg/l loading rate WAF. 

Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 and 48 hours (see details on analytical methods section) gave measured concentrations of 97.7 mg/l and 101 mg/l at 0 and 48 hours respectively. Only samples at the No Observed Effect Loading Rate (NOEL) and above were analysed.

Table1              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
10	0	0
100	0	0

Definitive Test Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours.

 

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
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

 

R₁– R₄= Replicates 1 to 4

There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:

24 hr EL50 (mg/l): >100

48 hr EL50 (mg/l): >100

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

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 (see Table 3).

Table 3              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Water Column (cm)	14.8	14.8	14.7	14.7
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

 

*= Start of mixing period

+= End of mixing period

Validation of Mixing Period

Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured levels of test item, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis purified water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 71 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis. 

The results are summarised as follows:

Test Item Loading Rate (mg/l)	Measured Concentration (mg/l)
	Time (hours)
	24	72
Control	<LOQ	<LOQ
100	59.3	82.9

Although the results from this pre-study investigational work showed that the measured concentration increased from 24 hours to 72 hours, chemical analysis of the 100 mg/l Loading rate WAF test samples from the range-finding test showed that 24 hours was a sufficientpreparation period to ensure the maximum amount of the dissolved water soluble fraction of the test item. The preparation of the WAF was maintained at 24 hours.

 LOQ = Limit of Quantitation (assessed down to 0.03 mg/l)

Physico-chemical measurements

The results of the physico-chemical measurements are given below.

Temperature was maintained at 21 to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Some of the temperatures were measured to be slightly in excess of the 20 ± 1°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 in the control daphnids throughout the duration of the

test and that the temperatures were within the test guideline specification.

Physico-Chemical Measurements

Nominal Loading Rate (mg/l)		0 Hours				24 Hours	48 Hours
		pH	mg O2/l	%ASV*	T°C	TºC	pH	mg O2/l	%ASV*	T°C
Control	R1	7.9	8.8	99	21	22	8.2	8.7	100	22
	R2	7.9	8.8	99	21	22	8.2	8.7	100	22
	R3	7.9	8.7	98	21	22	8.1	8.7	100	22
	R4	7.9	8.7	98	21	22	8.1	8.6	99	22
100	R1	7.6	8.7	98	21	22	8.0	8.4	97	22
	R2	7.5	8.6	97	21	22	7.9	8.4	97	22
	R3	7.5	8.6	97	21	22	7.8	8.4	97	22
	R4	7.5	8.6	97	21	22	7.7	8.4	97	22

 

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

R₁– R₄= Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.* EL = Effective Loading rate

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21 to 22ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EL*₅₀ for the test item to Daphnia magnab ased 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.

Information supplied by the Sponsor, indicated that the test item contained approximately 42% 2-ethylhexyl dihydrogen phosphate (MW = 210 g/mol) and 49% bis(2-ethylhexyl) hydrogen phosphate (MW = 322 g/mol)). Under the acidic conditions of the analysis, both compounds could be observed, but the lower acidity and greater response of the bis(2-ethylhexyl) hydrogen phosphate meant that it appeared as the main peak in the chromatogram.

Analysis of the test preparations at 0 hours showed measured concentrations of of 96.8 mg/l and 97.2 mg/l in replicates R1 and R2 of the 100 mg/l loading rate WAF respectively. Measured concentrations of 96.4 mg/l and 98.2 mg/l were observed in replicates R1 and R2 respectively of the 100 mg/l loading rate WAF at 48 hours.

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.

*EL = Effective Loading rate

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January to 2 February 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the principles of GLP and in accordance with an ISO testing guideline. Validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

other: ISO TC147/SC5/WG2

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The behaviour category of the test substance was cataegorised according to PARCOM as follows: There were floating and settled liquids or solids Therefore appropriate amounts of test substance were added to sea water while stirring (magnetic stirrer). The vessel was sealed and the contents stirred in the dark for 20-24 hours at a speed sufficient to suspend all floating and settled liquid or solid material. The mixture was allowed to stand for 4 hours phase-separation and a subnatant aqueous sample siphoned off, avoiding all settled and floating material, and used as test solution.

Test organisms (species):

other: Acartia tonsa

Details on test organisms:

Acartia tonsa (Dana), originally from laboratory cultures at the Laboratory of Marine Biology, 3000 Helsingar, Denmark, were cultured at the test facility. Eggs were hatched and stock cultures of Acartia tonsa were maintained in aerated seawater in 10 litre round, glass flasks at the test temperature of 20 ± 2 °C. Illumination is provided by cool, white fluorescent lights on a 12 h light, 12 h dark cycle. The culture was fed on cultured algal cells (Rhodomonas baltica).

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 2°C

pH:

3.8 to 8

Dissolved oxygen:

>4 mg/L

Salinity:

32%

Nominal and measured concentrations:

Nominal test concentrations: 103, 202, 502, 1011 and 2006 mg/L

Details on test conditions:

Four replicates were used for the water control and each concentration of the test substance. Test vessels consisted of 50 mL plastic beakers, each containig 25 mL of test solution.The test was conducted under a 12 h photoperiod.

Reference substance (positive control):

yes

Remarks:

1 mg/L 3,5-dichlorophenol

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1 410 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

See Table 1 for a summary of the mortality observations

Results with reference substance (positive control):

See Table 1

Table 1. Mortalty of Acartia tonsa exposed to OFLP 95/1820 in seawater

Concentration (mg/L)	Number of test animals per concentration	Number of dead animals at 24 h	Number of dead animals at 48 h
0 (Control)	21	0	1
103	20	0	0
202	21	0	0
502	20	0	0
1011	21	0	0
2006	21	21	21
Reference toxicant	20	11	15

Validity criteria fulfilled:

yes

Conclusions:

The 48 h LC50 was estimated to be 1410 mg OFLP 95/1820/L.

Executive summary:

An acute toxicity test was conducted in which the marine copepod Acartia tonsa was exposed to OFLP 95/1820. The 48 h LC50 was estimated to be 1410 mg OFLP 95/1820/L.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 3 and 11 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the principles of GLP and in accordance with an ISO testing guideline. Validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

other: ISO/CD 14669: Determination of Acute Lethal Toxicity to Marine Copepods

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: PARCOM Ring Test Protocol: Acute Toxicity to the Marine Copepod Acartia tonsa

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: Due to the low aqueous solubility and complex nature of the test item for the purposes of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).The test substance for each concentration was mixed with filtered and aerated seawater in a glass-flask with stopcock and stopper on a magnetic stirrer for -20 hours.The flask was wrrapped in aluminum foil to avoid light exposure and the top was covered with parafilm to minimize volatilization. After mixing, the solution was separated for 1-4 hours. The first 50 mL was discrded and the middle phase was collected for toxicity testing.- Eluate: Not applicable- Controls: A positive control of 1.0 mg/L 3,5-dichlorophenol was used.- Evidence of undissolved material : None given

Test organisms (species):

other aquatic arthropod: Acartia tonsa

Details on test organisms:

Acartia tonsa eggs used in the definitive test were obtained from the University of Copenhagen, Laboratory of marine biology, Helsingor, Denmark, on 19.07.01 and stored in the refrigerator. A synchronous culture was used. The animals were cultivated in small batches. The age of the animals in the test was 19 days.

Test type:

not specified

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19.8 to 20.6°C

Dissolved oxygen:

7.2 mg/L

Nominal and measured concentrations:

Nominal concentrations used were: 0, 100, 250, 500 and 1000 mg/L

Details on test conditions:

Four concentrations of the test substance, replicated four times were tested. Four replicate vessels were set up for the water control. Five test organisms were used per replicate vessel. Percent mortality was recorded after 24 and 48 hours.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol.

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

158.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

117.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

See Table 1 below

Results with reference substance (positive control):

- Mortality: 60% mortality after 48 h

Validity criteria fulfilled:

yes

Conclusions:

The 48 h LC50 for Acartia tonsa exposed to Servo RM 93700 was 117.4 mg/L

Executive summary:

An acute toxicity test was performed in which the marine organism Acartia tonsa were exposed to Servo RM 93700. The LC50 after 48 h was deterined to be 117.4 mg/L.

Description of key information

48h EL50 = >100 mg/L (Daphnia magna); Goodband & Mullee (2011); OECD 202
48h LL50 = 117.4 mg/L (Acartia tonsa); Furst (2002); ISO/CD 14669
48h LL50 = 1410 mg/L (Acartia tonsa); Torp (1996); ISO TC147/SC5/WG2

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

117.4 mg/L

Additional information

Three key studies are submitted for the short-term toxicity to aquatic invertebrates endpoint, one for a freshwater species and two for marine species. Each study was conducted using a standard approach for testing poorly soluble, multi-component substances, the preparation of a water accommodated fraction (WAF). As such, the effect values are presented as loading rates (e.g. LL₅₀or EL₅₀), and not measured aqueous concentrations.