Phosphinylidynetrimethanol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-10 - 2018-04-12 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

"OECD guidelines for testing of chemicals 202: Daphnia sp. Acute Immobilisation Test" (2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

UHPLC-am-MS was used for identification of THPO: 5 µL samples were directly injected into the MS. During the test triplicate samples (10 ml) were collected from the freshly prepared blank solution and the highest and lowest test concentrations, and again samples were taken from all 3 solutions at the end of the test from the test vessels.
Samples were stored in glass vials at -20 °C until measurement.
High-resolution ICP-MS was used to quantify THPO: samples (5ml) were collected in parallel to those for the UHPLC measurements from the same solutions at the start and the end of the test. These samples were collected in polyethylene tubes containing HNO3 (final concentration 2 %). For the measurements, the test samples were diluted 1/10 or 1/100 in 2 % HNO3.

Vehicle:

yes

Remarks:

OECD medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: OECD medium was used as a control and dilution medium; 200.2 mg THPO was weighed and added to 200 ml of OECD medium. The test item is easily soluble in the medium.
- Controls: ZnCl2 was tested as reference item, OECD medium was used as the dilution medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test item is easily soluble in the medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: DM 04 02 K36
- Source: Test organisms are grown at the VITO laboratory facility.
- Feeding during test: no

ACCLIMATION
- Acclimation period: As required by OECD guideline 202 a daphnia breeding subculture in OECD medium was started on 19/03/2018 (3 weeks before test). The health condition of this new culture was considered as normal: normal life cycle rhythm, average mortality rate, average number of youngsters

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For the test neonate organisms (max. 24 hours of age) were used. On 09/04/2018 (one day before test start) some parent organisms were separated in a breeding vessel and fed with enriched algae food. The youngsters that were born overnight were then used for this test. Survival in control conditions during the test was 100% indicating that breeding conditions did not cause acute effects. The sensitivity for the reference item ZnCl2 of organisms from the OECD medium breeding compartment was significantly higher compared to the JP4 cultured organisms.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Hardness:

no details given

Test temperature:

19.1 - 20.8 °C

pH:

7.76 - 8.19

Dissolved oxygen:

90 - 99 %

Salinity:

no details given

Conductivity:

142 - 152 µS/cm

Nominal and measured concentrations:

Results showed that recovery at the end of the test is > 80 % of the initial values for both analytical methods.
The HPLC-MS data indicate a full recovery of the 3 masses at the highest concentration, indicating that no degradation products are generated.
It is concluded that the test item is stable (not degrading) during the test. As P recovery at the end is 81 % of the initial value, it is concluded that the test item is stable during the test and test concentrations are within the nominal range.
Nominal concentrations for the test: 100 - 50 - 25 - 12.5 - 6.25 mg/l THPO.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml glass vessels, with transparent lid
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume of 20 ml per vessel
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: Light/Dark= 16h/8h
- Light intensity: not critical

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 24 and 48 hours of exposure the mobility of the organisms was evaluated: the vessels were slightly shaken to provoke movement of the organisms, and the number of mobile organisms was registered.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: A preliminary range-finding test (DAC 18 007; 1/10 dilutions in JP4 medium) showed an EC50 in the range between 10 and 100 mg/l. It was decided to use the next test concentrations for the final test: 100 - 50 - 25 - 12.5 - 6.25 mg/l THPO.

Reference substance (positive control):

yes

Remarks:

Periodically an acute daphnia test is performed with ZnCl2 as a reference item. The last test was performed in February 2018.

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No, all test organisms were active and mobile after 48 hours of exposure at the test concentration of 100 mg/L.
- Mortality of control: No, mean survival in the control replicates was 100%.
- Other adverse effects control: No.
- Abnormal responses: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Relevant effect levels:
- OECD medium was used as the dilution medium in this test. The EC50 (24h) = 6 mg/l ZnCl2 and EC50 (48h) = 0.29 mg/l ZnCl2.
- Results in standard JP4 medium: historical range for EC50 (48h) = 1.04-2.35 mg/L ZnCl2, mean value 1.7 mg/L ZnCl2
- ECx: EC50 (48h) = 2.2 mg/L ZnCl2
It was shown that the sensitivity of Daphnia to ZnCl2 toxicity was significantly higher in the OECD cultured organisms, that were used in this study.

Reported statistics and error estimates:

A statistical analysis was not necessary, as no immobilisation or any other adverse effects were observed.

Analytical data

Results showed that recovery at the end of the test is > 80 % of the initial values for both analytical methods.

Based on the composition of the medium the expected P concentration in blank OECD medium is 360 µg/l P. In the test dilutions 1660, and 22360 µg/l P are expected in the 6.25 and 100 mg/l THPO dilutions respectively (THPO = C3H9O4P = > 22 % P). The measured values at the start of the test are within the expected range (at least 89 % of the nominal value). At the end of the test, the P concentration in the 100 mg/l THPO dilution is decreased to 72.5 % of the expected value, but to 81% of the initial value. The HPLC-MS data indicate a full recovery of the 3 masses at the highest concentration, indicating that no degradation products are generated.

Validity of the test

Validity criteria were met:

• Mean survival in the control replicates was 97 % and met the validity criterion that not more than 10 % of the daphnids should be immobilised.
• The dissolved oxygen concentration was ≥ 96 % oxygen in all test conditions and met the validity criterion of at least 3 mg/l (appr. 30 % of the air saturation at 20°C) during the test duration.

Test results

No significant mortality was seen after 24 h and 48 h of exposure. All test organisms were active and mobile after 48 hours of exposure at the test concentration (100 mg/L) and LC50 > 100 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

LC50 (48h): > 100 mg/l (nominal)

Executive summary:

The study was conducted under GLP to evaluate the effect of the presence of the test item Tris(hydroxymethyl)phosphine oxide (THPO) on the mobility of Daphnia magna. The acute toxicity test is based on the OECD test protocol "OECD guidelines for testing of chemicals 202: Daphnia sp. Acute Immobilisation Test" (2004).

Based on preliminary test results the nominal test concentrations 100 - 50 -25 - 12.5 - 6.25 mg/l Tris(hydroxymethyl)phosphine oxide were used. Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted. None of the concentrations showed toxicity and also none of the animals was immobilised in the blank control.

ZnCl2 was tested as reference item in a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test item in the test solution was determined using UHPLC-am-MS to demonstrate the integrity of the molecules during the test (qualitative evaluation), and phosphorous (P) is analysed by ICP-AES to quantify the THPO concentration. Results showed that recovery at the end of the test is ≤ 80 % of the initial values for both analytical methods

Exposure concentrations up to 100 mg/l (analytical concentration) of the test item Tris(hydroxymethyl)phosphine oxide (THPO) for 48 hours had no adverse effect on the mobility of Daphnia. It is concluded that the LC50 is >100 mg/l.

Description of key information

LC50 (48h) for freshwater invertebrates (Daphnia): >100 mg/l  [nominal; based on mobility; OECD 202]

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A GLP study was conducted to evaluate the effect of the presence of the test item Tris(hydroxymethyl)phosphine oxide (THPO) on the mobility of Daphnia magna. The acute toxicity test is based on the OECD test protocol "OECD guidelines for testing of chemicals 202: Daphnia sp. Acute Immobilisation Test" (2004). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on preliminary test results, the nominal test concentrations 100 - 50 -25 - 12.5 - 6.25 mg/l Tris(hydroxymethyl)phosphine oxide were used. Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted. None of the concentrations showed toxicity and also none of the animals was immobilised in the blank control.

ZnCl2 was tested as reference itemin a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test item in the test solution was determined using UHPLC-am-MS to demonstrate the integrity of the molecules during the test (qualitative evaluation), and phosphorous (P) is analysed by ICP-AES to quantify the THPO concentration. Results showed that recovery at the end of the test is ≤ 80 % of the initial values for both analytical methods

Exposure concentrations up to 100 mg/l (analytical concentration) of the test item Tris(hydroxymethyl)phosphine oxide (THPO) for 48 hours had no adverse effect on the mobility of Daphnia. It is concluded that the LC50 is >100 mg/l.