bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is highly insoluble in water, hence indicating that aquatic toxicity is unlikely to occur

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VII column 1, 9.1.1. Short-term toxicity testing on invertebrates, The registrant may consider long-term toxicity testing instead of short-term. According to column 2, 9.1.3. The study does not need to be conducted if:
— there are mitigating factors indicating that aquatic toxicity is unlikely to occur, for instance if the substance is highly insoluble in water or the substance is unlikely to cross biological membranes, or — a long-term aquatic toxicity study on fish is available. Long-term aquatic toxicity testing as described in Annex IX shall be considered if the chemical safety assessment according to Annex I indicates the need to investigate further effects on aquatic organisms. The choice of the appropriate test(s) will depend on the results of the chemical safety assessment.
The long-term aquatic toxicity study on fish (Annex IX, Section 9.1.6) shall be considered if the substance is poorly water soluble.

In general, there is no legal limit value available defining a substance as poorly water soluble or insoluble under REACH. However, in ECHA’s Guidance document Chapter R.7b: Endpoint specific guidance Version 3.0 – February 2016, it is stated: „poorly water soluble substances (e.g. water solubility below 1 mg/L or below the detection limit of the analytical method of the test substance)“. Further, poorly soluble substances are defined by OECD (2000 OECD SERIES ON TESTING AND ASSESSMENT, Number 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, ENV/JM/MONO(2000)6) as substances with a limit of solubility <100 mg/l although technical problems are more likely to occur at <1mg/l as defined in TGD (1996). Very low water solubility (i.e. in the low μg/l range) could be used as a reason to significantly modify a standard test or to test non-pelagic organisms preferentially.
ECHA’s Guidance document Chapter R.7c: Endpoint specific guidance Version 3.0 – June 2017 further says: „As indicated in the OECD TG 305, for strongly hydrophobic substances (log Kow > 5 and a water solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. However, an aqueous exposure test is preferred for substances that have a high log Kow but still appreciable water solubility with respect to the sensitivity of available analytical techniques, and for which the maintenance of the aqueous concentration as well as the analysis of these concentrations do not pose any constraints.
The logPow was determined to be >7.2, and the water solubility is < 4.5 µg/L, so the criteria mentioned above are met. Further, the criterion for “below the detection limit of the analytical method of the test substance” is also met. The establishment and validation of the analytical method was properly performed.
The limit of quantification (LOQ) is defined as the lowest concentration level at which an accuracy in the range 70-110% and a repeatability of ≤ 20% is demonstrated. The LOQ was obtained from the data of the accuracy and repeatability test. The limit of quantification (LOQ) was assessed at a test item concentration of 10 µg/L in water.
The calibration curve was constructed excluding the data points in the concentration range 0.0400 - 0.250 µg/L since no reliable duplicate results and no linear or quadratic relationship between response of disulfide (n=0) and test item concentration was observed.
In the concentration range 0.450 – 5.00 µg/L, the calibration curve was constructed excluding one data point since a deviation was observed in the response factor. There was a quadratic relationship between response of disulfide (n=0) and test item concentration in the range of 0.450 – 5.00 µg/L (in end solution). Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies of the remaining data points were in the range 85-115% the calibration line was accepted. Pretreatment of the samples however required a dilution by a factor of 10 with 2-propanol, which does not allow a quantification in these low limits.
Ecotoxicity tests are performed in media containing various supplements and biological material, which is expected to lower the sensitivity of the analytical method. Hence, quantification of the test item up to its water solubility in ecotoxicity test media could like not be performed at all. So, the required analytics for ecotoxicity testing, both short and long-term, are technically not feasible, and testing does not need to be conducted.
Based on the available information it can be stated that, as outlined in the first waiving possibility under REACH, the substance is highly insoluble in water, and hence, aquatic toxicity is unlikely to occur. The latter conclusion is supported by available studies conducted prior to REACH implementation, i.e. a study for toxicity against Brachydanio rerio. In this study, the following LC-values (Mortality, effective concentrations) were determined: LC100 = 12.0 mg/L; LC50 = 2.38 mg/L; LC0 = 0.64 mg/L; NOEC = 0.64 mg/L, which are way above the possible water solubility of the registered substance. With regard to fish, adequate information for environmental classification and labelling is available, as the noted effects were above the water solubility and trigger classification as Aquatic Chronic Cat. 4, so the second waiving criterion under REACH is also met, as the assessment of the available data does so not indicate the need to investigate further effects on aquatic organisms.

Hence, an additional study of the acute toxicity of the registered substance against fish does not need to be conducted, as it is considered both not technically feasible and no additional information on classification and labelling could be retrieved, and the study can be omitted due to animal welfare.

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

water solubility

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996-01-15 - 1996-01-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

determination of the actual concentration not sufficiently reliable taking into account recent water solubility determinations

Reason / purpose for cross-reference:

reference to other study

Remarks:

Water solubility

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

as of 1992

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry and dark storage in closed vessels at room temperature

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1/1 saturated solution, 1/2 saturated solution, 1/4 saturated solution, 1/8 saturated solution, 1/16 saturated solution
- Sampling method: Water samples (500 ml each) of the different test item concentrations were taken at the stipulated times (0 h -new and 24 h - old, 72 h - new and 96 h - old).
- Sample storage conditions before analysis: 2 ml methanol were added to each sample and the samples were frozen immediately. They were analysed together after the fish test was finished.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is virtually insoluble in potable water (as reported by the sponsor). Saturated solutions are made and diluted to lower saturation levels.
Each pool contained 10 l dilution water.
0.5 ml test item was transferred into a 5000 ml volumetric flask and filled up to volume with dilution water. This was done twice (1st and 2nd solution). These two solutions were stirred overnight for the purpose of saturation. Afterwards both solutions were filtrated into pool No. 2. Resulting concentration: saturated solution of the test item.
0.5 ml test item was transferred into a 5000 ml volumetric flask and filled up to volume with dilution water (3rd). This solution was stirred overnight for the purpose of saturation. Subsequently this solution was filtrated into pool No. 3 containing 5000 ml dilution water. Resulting concentration: 1/2 saturated solution.
0.5 ml was transferred into a 5000 ml volumetric flask and filled up to volume with dilution water (4th). This solution was stirred overnight for the purpose of saturation. Then 2500 ml of this solution was filtrated into pool No. 4 containing 7500 ml dilution water. Resulting concentration: 1/4 saturated solution.
1250 ml of the 4th saturated solution made before was filtrated into pool No. 5 containing 8750 ml dilution water. Resulting concentration: 1/8 saturated solution.
625 ml of the 4th saturated solution made before was filtrated into pool No. 6 containing 9375 ml dilution water. Resulting concentration: 1/16 saturated solution.
The saturated solutions and the dilution water as well were prepared the day before (in the afternoon). They were stirred overnight in the same cabinet and at the same temperature as the fish test was performed.

- Controls: The control pool (pool No. 1) contained dilution water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): To all appearances not dissolved test item was not discernible in the pools except for the saturated and half saturated test item solutions of the last water renewal which appeared cloudy.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish (Danio rerio)
- Source: Südharz-Aquarium GmbH, Scharzfelder Str. 107, D-37431 Bad Lauterberg, Germany
- Length at study initiation (length definition, mean, range and SD): 2.5 - 3.5 cm

ACCLIMATION
- Acclimation period: The test fish were acclimatized at least 12 -15 days.
- Acclimation conditions (same as test or not): During the acclimatization period the fish were not treated. At least 7 days the test fish were kept in water of the quality used during the test.
- Type and amount of food during acclimation: Tetra Min
- Feeding frequency during acclimation: Daily and discontinued at least 24 hours prior to the test period.
- Health during acclimation (any mortality observed): The test fish were in good health. One fish of 150 died.

FEEDING DURING TEST
- Amount: no feeding during the test period

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

Derived from the CaCl2 addition, the hardness of water was equal to 2 mmol/l CaC03 which corresponds to a hardness of 200 mg/l CaC03 (valid: 10 - 250 mg/l)

Test temperature:

22.1 - 22.5 °C (water temperature)

pH:

7.29 - 7.95 in the new (freshly prepared) test solutions
7.39 - 7.87 in the old (24 h) test solutions

Dissolved oxygen:

8.2 - 8.6 in the new (freshly prepared) test solutions
8.0 - 8.5 in the old (24 h) test solutions

Conductivity:

0.4 -1.5 µS/cm

Nominal and measured concentrations:

Nominal: control, 1/1 saturated solution, 1/2 saturated solution, 1/4 saturated solution, 1/8 saturated solution, 1/16 saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: pools
- Material, size, headspace, fill volume: 10 l volume
- Aeration: yes, with aeration pumps (type SP 402 SLR)
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: maximum loading of 1.0 g fish/litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Conductivity of deionized water: 0.4 -1.5 µS/cm
Dilution water which was used in the test period was reconstituted water:
CaCl2 2 mmol/l
MgS04 0.5 mmol/l
NaHC03 0.75 mmol/l
KC1 0.075 mmol/l
The sum of calcium and magnesium ions in the dilution water was 2.5 mmol/l. The proportion of Ca : Mg ions was 4 : 1 and of Na : K ions 10 : 1. The acid capacity Ka43 of the solution was 0.8 mmol/l.
Derived from the CaCl2 addition, the hardness of water was equal to 2 mmol/l CaC03 which corresponds to a hardness of 200 mg/l CaC03 (valid: 10 - 250 mg/l).
pH values of test waters: neutral (7.3 - 7.8)
For each water renewal the reconstituted water was left ready prepared overnight.

- Culture medium different from test medium: yes

Tap water of Karlsruhe with the following quality was used during holding time and during the first period of acclimatization:
acid capacity Ka 43: 4.75 - 4.77 mmol/l
sum of calcium and magnesium ions: 2.83 - 3.14 mmol/l
calcium Ca2+: 2.41 - 2.60 mmol/l
magnesium Mg2+: 0.42 - 0.54 mmol/l
sodium Na + : 0.28 - 0.33 mmol/l
potassium K+: 0.05 - 0.06 mmol/l
pH: 7.39-7.49

- Intervals of water quality measurement: The dissolved oxygen concentrations and the pH values of the control and of the test solutions were checked daily at 24 hours intervals

OTHER TEST CONDITIONS
- Adjustment of pH: The pH values of the test solutions were not adjusted.
- Photoperiod: Light-dark-cycle: 22 ± 1 °C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The fish were inspected at least daily for mortality and any visible signs of abnormality during the test time of 96 hours. Fish were considered to be dead if touching the caudal peduncle produced no reaction. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: control, 1/1 saturated solution, 1/2 saturated solution, 1/4 saturated solution, 1/8 saturated solution, 1/16 saturated solution

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.38 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

Value calculated using the saturation level of 27% (concentration which proved lethal to 50 % of fish at the time of 96 h, derived graphically) and the water solubility analytically determined to be 8.8 mg/l for the test item during analytical validation.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other:

Remarks:

measured concentrations of the test item in the aged (24 h old) test solutions after 96 h

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other:

Remarks:

measured concentrations of the test item in the aged (24 h old) test solutions after 96 h

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other:

Remarks:

measured concentrations of the test item in the aged (24 h old) test solutions after 96 h

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
24 h: 1/1 saturated solution the test item: 1 fish shows loss of equilibrium.
48 h: 1/4 saturated solution: 1 fish shows loss of equilibrium.
72 h: 1/8 saturated solution: 1 fish shows loss of equilibrium.
96 h: 1/2 saturated solution: 1 fish swims slowly and shows loss of equilibrium.
96 h: 1/4 saturated solution: 1 fish swims slowly and shows loss of equilibrium.
At any other time of inspection no visible signs of abnormalities of surviving test fish were observed.
- Observations on body length and weight:
total length of 10 fish: sample 1: mean 2.8 cm (standard deviation 0.254 cm), sample 2: mean 2.9 cm (standard deviation 0.135 cm)
total weight of 10 fish: sample 1: mean 0.19 g (standard deviation 0.068 g), sample 2: mean 0.20 g (standard deviation 0.028 g)
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: none stated
- Effect concentrations exceeding solubility of substance in test medium: In the aged (24 h old) test medium of the 1/1 saturated solution 12.0 mg test item/L were found after 96 hours, exceeding the water solubility of 8.8 mg/l analytically determined during analytical validation.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

Statistical method:
The LC 50 value (72, 96 h) was graphically obtained resulting from the plot on probit paper (see "Attached background material").

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP in a semi-static 96 hours test according to EU method C.1 on the registered substance itself with no relevant deviations. Thus, the results were obtained via a scientifically reasonable method. Due to the poor water solubility of the test substance, the adjustment of precise concentrations was difficult. Therefore, saturated solutions were prepared and diluted to lower saturation levels, resulting in 1/1, 1/2, 1/4, 1/8 and 1/16 saturated solutions. These solutions were used as test concentrations and determined analytically by HPLC-UV detecition. Hence, the results can be considered as reliable to assess the effects of the test item to aquatic organisms, here, zebra fish.
The following LC-values (effective concentrations) were determined accordingly:
Mortality
LC100 = 12.0 mg/L
LC50 = 2.38 mg/L
LC0 = 0.64 mg/L
NOEC = 0.64 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, the test item should be classified as aquatic chronic Cat. 4, taking into account the facts that the test item is actually insoluble in water, i.e. <4.5 µg/l as shown in a recent water solubility test, and not readily biodegradable. Based on the given data it is not absolutely evident what is the basis for the effects, as the above-mentioned concentrations are above the water solubility. Either the batch which was used for the test contained a higher amount of water-soluble impurities, or the stated concentrations underestimate the actual LC50 and the substance is already toxic at lower concentrations, or the lethal effects are based on mechanical effects resulting from undissolved test material, e.g. adhering of the gills.
According to Regulation 1272/2008, ‘Safety net’ classification, Category Chronic 4, is adequate in cases when data do not allow classification under the above criteria but there are nevertheless some grounds for concern. This includes, for example, poorly soluble substances for which no acute toxicity is recorded at levels up to the water solubility (note 4), and which are not rapidly degradable in accordance with section 4.1.2.9.5 and have an experimentally determined BCF ≥ 500 (or, if absent, a log Kow ≥ 4). Even though toxic effects occurred, the nominal or therein measured effect values are higher (or unknown) than the recently determined water solubility of the substance, so classification as Chronic 4 is considered adequate.

Executive summary:

In a 96-h acute toxicity study, zebra fish (Brachydanio rerio) were exposed to bis (0,0-2-ethylhexyl-thiophosphoryl)polysuIfide at different concentrations under semi-static conditions.  Due to the poor water solubility of the test substance, the adjustment of precise concentrations was difficult. Therefore, saturated solutions were prepared and diluted to lower saturation levels, resulting in 1/1, 1/2, 1/4, 1/8 and 1/16 saturated solutions. These solutions were used as test concentrations and determined analytically by HPLC-UV detection. The 96-h LC₅₀ was graphically obtained resulting from the plot on probit paper and was determined to be 2.38 mg/L.  The LC0 and NOEC values, based on mortality/sublethal effects, were 0.64 mg/L, respectively, based on measured concentrations of the test item in the aged test solutions (24 h old) after 96 hours of test duration.  Sublethal effects like slow swimming and loss of equilibrium were observed on one fish only in the groups exposed to the 1/1, 1/2, 1/4 and 1/8 saturation solution, respectively, at different observation times and were therefore considered to be negligible. 

Based on the results of this study, the test item would be classified as aquatic chronic Category 4 to zebra fish (Brachydanio rerio) according to Regulation 1272/2008 and amendments.

Description of key information

Short-term toxicity to fish: LC100 (96h) = 12.0 mg/L, LC50 (96h) = 2.38 mg/L, LC0 (96h) = 0.64 mg/L, NOEC (96h) = 0.64 mg/L for zebra fish (Brachydanio rerio) (EC Directive 92/69/EEC, Method C.1, semi-static)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.38 mg/L

Additional information

Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, the test item should be classified as aquatic chronic Cat. 4, taking into account the facts that the test item is actually insoluble in water, i.e. <4.5 µg/l as shown in a recent water solubility test, and not readily biodegradable. So actually the available LC50 is not considered to suffice as a basis for chemical safety assessment.