Phosphinylidynetrimethanol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-05-11 - 2020-08-19 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute Toxicity Test", June 18, 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document, Series on Testing and Assessment No. 126, “Short Guidance in the Threshold Approach for Acute Fish Toxicity”, May 31, 2010

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 144 mg test item/L (equivalent to 100 mg THPO/L based on a purity of 69.47 %) and a control
- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken on day 0 and day 3 of exposure.
For the determination of the stability of the test item and the maintenance of the test item concentrations under the test conditions, respectively, duplicate samples from the aged test media of the only one test concentration were taken at the first test medium renewal (on day 1) and at the end of the last renewal period (on day 4).
All test samples were taken from the approximate centre of the aquaria.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the only test item concentration of nominal 144 mg/L (equivalent to 100 mg THPO/L based on a purity of 69.47 %) was prepared by dissolving 1728.3, 1728.0, 1728.8 and 1728.4 mg test item into 12002.1, 12000, 12005.5 and 12002.8 mL test water by intense stirring for 10 minutes.
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test media were observed daily. There were no remarkable observations.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: The test fish were obtained from Mohnen aquaculture, Teichanlage Schevenhütte, Nideggener Strasse 124-128 and 150, 52224 Stolberg, Germany.
- Age at study initiation: Juveniles
- Length at study initiation (length definition, mean, range and SD): The mean body length of the fish in the test was 5.07 cm ± 0.2 cm (Mean ± SD).
- Weight at study initiation (mean and range, SD): The mean body wet weight of the fish in the test was 1.32 g ± 0.1 g (Mean ± SD).
- Maintenance of the brood fish: All fish were obtained and held in the laboratory for at least 9 days before the start of the test. They were held in water of the quality to be used in the test for at least seven days immediately before testing under the following conditions:
Light: 12 hours photoperiod daily
Temperature: 10 - 14 °C
Oxygen concentration: at least 80 % of the air saturation value
Feeding: three times per week or daily until 24 hours before the test was started
During the last 7 days prior to the start of the test no fish (0 %) died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.

ACCLIMATION
- Acclimation period: Not necessary since the fish were held in water of the quality to be used in the test for at least seven days immediately before testing.

FEEDING DURING TEST
- Amount: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Water Hardness: 1.78 mmol/L (= 178.4 mg/L) as CaCO3

Test temperature:

12.1 to 13.8 °C

pH:

7.2 to 7.8

Dissolved oxygen:

92 to 100 % of the air saturation value

Nominal and measured concentrations:

nominal: 144 mg test item/L (equivalent to 100 mg THPO/L based on a purity of 69.47 %) and a control
measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: 16 litre glass aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 12 litre test medium
- Aeration: The test media were slightly aerated during the test.
- Renewal rate of test solution (frequency/flow rate): A semi static test with test medium renewal every day was chosen to keep the concentration of the test item in the test media as constant as possible during the test period of 96 hours. During this semi static test the surviving fish were placed in a clean aquarium with freshly prepared test medium of the corresponding concentration every day
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.769 fish/L test water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (ISO Medium). For detailed information on the composition of the test water please refer to section "Any other information on materials and methods, including tables".
- Ca/mg ratio: 4 : 1 (based on molarity)
- Culture medium different from test medium: All fish were held in water of the quality to be used in the test for at least seven days immediately before testing.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 12 h light : 12 h dark (30 min dawn/dusk period was provided)
- Light intensity: 600 to 700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed at test start and after approximately 2, 4, 24, 29, 48, 53, 72, 77 and 96 hours of test duration for sublethal effects and mortality.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: The lowest EC50 value of existing relevant algae or acute invertebrate (e.g. daphnia) tests was set as threshold concentration. This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration or at 100 mg (active ingredient)/L.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%.

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

> 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 144 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The test concentration of 144 mg test item/L is equivalent to 100 mg THPO/L based on a purity of 69.47%

Details on results:

- Behavioural abnormalities: All fish showed no sublethal effects or behavioural abnormalities during the exposure time.
- Mortality of control: In the control no fish died during the test.
- Other adverse effects control: No.
- Effect concentrations exceeding solubility of substance in test medium: No. The test media were observed daily. There were no remarkable observations.

Reported statistics and error estimates:

No statistical analysis was performed. The LC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

LC50 (96h): >100 mg/l (nominal)

Executive summary:

The acute toxicity of Tris(hydroxymethyl)phosphine oxide (THPO) to fish was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test under GLP.

For this purpose, juvenile Rainbow Trout (Oncorhynchus mykiss) were exposed in a semi static test to the only concentration of nominal 144 mg test item/L (equivalent to 100 mg THPO/L) under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. A threshold approach was pursued.

The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species.

This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration or at 100 mg (active ingredient)/L. The lowest EC₅₀ value of existing relevant algae or acute invertebrate (e.g. daphnia) tests is set as threshold concentration.

In the interest of animal welfare and efficient use of resources, it is important to avoid the unnecessary use of animals whenever possible. In the field of aquatic toxicology, this especially applies to the acute toxicity testing of fish according to OECD 203. The threshold approach described hereafter addresses fish toxicity by using a single-concentration test (limit test) requiring less fish compared to the full acute fish toxicity study. It is based on the derivation of a threshold concentration from relevant algae and acute invertebrate (e.g. daphnia) tests at which fish toxicity is tested. If no mortality occurs in the limit test using the threshold concentration, the threshold concentration might be used as a surrogate LC₅₀ value in the further hazard or risk assessment. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

The quantification of the test item THPO in the test samples was performed using liquid chromatography with MS/MS. In the freshly prepared test media at the start of the test and at test medium renewal on day 3 a mean of 104 % of the nominal test concentration was found. In the aged test media after 24 hours of test duration and at day 4, a mean of 106 % of the nominal value was determined. During the test the test organism were exposed to a mean of 105 % of nominal.

In the control and the only test concentration of nominal 144 mg test item/L (equivalent to 100 mg THPO/L) all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. Based on these results the 96-hour LC₅₀ was determined to be > 144 mg test item/L (> 100 mg THPO/L) based on the nominal concentration. The NOEC was determined to be ≥ 144 mg test item/L (≥ 100 mg THPO/L) also based on the nominal concentrations. The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part. Since the concentration of the test item were within± 20 % of the nominal concentrations during the test all reported results refer to nominal values.

Description of key information

LC₅₀(96h) for freshwater fish (Oncorhynchus mykiss): >100 mg/l [nominal; based on mortality; OECD 203]

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The acute toxicity of Tris(hydroxymethyl)phosphine oxide (THPO) to fish was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test under GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Juvenile Rainbow Trout (Oncorhynchus mykiss) were exposed in a semi static test to the only concentration of nominal 144 mg test item/L (equivalent to 100 mg THPO/L) under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. A threshold approach was pursued.

The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species.

This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration or at 100 mg (active ingredient)/L. The lowest EC₅₀value of existing relevant algae or acute invertebrate (e.g. daphnia) tests is set as threshold concentration.

In the interest of animal welfare and efficient use of resources, it is important to avoid the unnecessary use of animals whenever possible. In the field of aquatic toxicology, this especially applies to the acute toxicity testing of fish according to OECD 203. The threshold approach described hereafter addresses fish toxicity by using a single-concentration test (limit test) requiring less fish compared to the full acute fish toxicity study. It is based on the derivation of a threshold concentration from relevant algae and acute invertebrate (e.g. daphnia) tests at which fish toxicity is tested. If no mortality occurs in the limit test using the threshold concentration, the threshold concentration might be used as a surrogate LC₅₀value in the further hazard or risk assessment. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

The quantification of the test item THPO in the test samples was performed using liquid chromatography with MS/MS. In the freshly prepared test media at the start of the test and at test medium renewal on day 3 a mean of 104 % of the nominal test concentration was found. In the aged test media after 24 hours of test duration and at day 4, a mean of 106 % of the nominal value was determined. During the test the test organism were exposed to a mean of 105 % of nominal.

In the control and the only test concentration of nominal 144 mg test item/L (equivalent to 100 mg THPO/L) all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. Based on these results the 96-hour LC₅₀was determined to be > 144 mg test item/L (> 100 mg THPO/L) based on the nominal concentration. The NOEC was determined to be ≥ 144 mg test item/L (≥ 100 mg THPO/L) also based on the nominal concentrations. The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part. Since the concentration of the test item were within ± 20 % of the nominal concentrations during the test all reported results refer to nominal values.