Diphenyl methylphosphonate

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:

2013-04-22 - 2013-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of The United Kingdom

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations and stability of the test substance: at 0 and 48 h
Water temperature and light intensity: recorded daily
Dissolved oxygen concentration and pH: start and end of the experiment
Observation of immobilisation/adverse reactions: at 24 and 48 h

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
Prior to use, the test substance was melted at approx. 50 °C. 100 mg of it was dissolved in reconstituted water with the aid of ultrasonication for 30 min and the volume adjusted to 1 L to give the 100 mg/L stock solution. Aliquots (10, 18, 32, 56 and 100 mL) were each separately added to 1 L of reconstituted water to give the test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L respectively. Each stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea
- Strain: 1st instar
- Source: in-house laboratory culture
- Age at study initiation: < 24 h
- Feeding during test: no
- Food type (before): algal suspension (Desmodesmus subspicatus) and Tetramin(R) flake food suspension
- Frequency: daily
- Maintenance: at approx. 20 °C in 150 mL glass beakers containing Elendt M7 medium with a light cycle of 16/8 h light/darkness with 20 min dawn and dusk transition periods
- Method of reproduction: parthenogenesis

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period described.

Hardness:

250 mg/L CaCO3

Test temperature:

20 - 21 °C

pH:

8.0 - 8.1

Dissolved oxygen:

8.7 - 9.2 mg O2/L
98 - 101 % of Air Saturation Value (ASV)

Salinity:

Not applicable.

Nominal and measured concentrations:

1.0, 1.8, 3.2, 5.6 and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- Fill volume: 200 mL
- Type: closed
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

RECONSTITUTED WATER
- Stock solutions: a) 11.76 mg/L CaCl2 x 2 H2O; b) 4.93 mg/L MgSo4 x 7 H2O; c) 2.59 mg/L NaHCO3; 0.23 mg/L KCl
- Preparation: 25 mL of each solutions a - d was added to each liter (final volume) of deionized water with a conductivity < 5 µS/cm. 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 approx. air-saturation value.

ELENDT M7 MEDIUM (Stock solution A)
- Constituents: 57190 mg/L H3BO3, 7210 mg/L MnCl2 x 4 H2O, 6120 mg/L LiCl, 1420 mg/L RbCl, 3040 mg/L SrCl2 x 6 H2O, 320 mg/L NaBr, 1260 Na2MoO4 x 2 H2O, 335 mg/L CuCl2 x 2 H2O, 260 mg/L ZnCl2, 200 mg/L CoCl2 x 6 H2O, 65 mg/L KI, 43.8 mg/L Na2SeO3, 11.5 mg/L NH4VO3, 5000 mg/L Na2EDTA x 2 H2O, 1991 mg/L FeSO4 x 7 H2O
- Preparation: an aliquot (depending on the volume of medium required) of each stock solution was added to a final volume of deionized reverse osmosis water to give stock solution A and stored at approx. 21 °C.

MACRO NUTRIENT STOCK SOLUTIONS
- Constituents: 293.80 g/L CaCl2 x 2 H2O (XV), 64.80 g/L NaHCO3 (XVI), 246.60 g/L MgSO4 x 7 H2O (XVII), 50.00 g/L Na2SiO3 x 9 H2O (XVIII), 58.00 g/L KCl (XIX), 2.74 g/L NaNO3 (XX), 1.84 g/L K2HPO4 (XXI), 1.43 g/L KH2PO4 (XXII)
- Vitamin nutrients: 750 mg/L Thiamine hydrochloride (XXIII), 10 mg/L Cyanocobalamine (Vitamin B12), 7.5 mg/L D(+)biotim (Vitamin H)
- Preparation: The final medium was prepared by adding an aliquot of Stock solution A (Elendt M7 Medium) along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the Vitamin Nutrient to the required amount (final volume) of deionized reverse osmosis water.
The pH of the prepared media was 8.0 +/- 0.2 and stored at approx. 21 °C.

OTHER TEST CONDITIONS
- Adjustment of pH: no - not required according to the applied guideline
- Photoperiod: 16 h light, 8 h darkness, 20 min dusk and down transition periods
- Light intensity: 473 - 501 lux

RANGE FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Immobilisation was observed at test concentrations of 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 2.6 - 3.3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 1.0 - 1.8

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The test preparations were observed to be clear colourless solutions during the exposure period.
The No Observed Effect Concentrations after 24 and 48 h were 1.8 and 1.0 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 h exposure were 3.2 and 1.8 mg/L respectively.

Results with reference substance (positive control):

EC50(24h): 1.0 mg/L (95 % Confidence limit: 0.91 - 1.20) / NOEC(24h): 0.56 mg/L / LOEC(24h): 1.0 mg/L
EC50(24h): 0.71 mg/L (95 % Confidence limit: 0.65 - 0.76) / NOEC(48h): 0.32 mg/L / LOEC(48h): 0.56 mg/L

Reported statistics and error estimates:

Trimmed Spearman-Karber method (Hamilton et al., 1977)

Table 2. Cumulative immobilization data in the Range-finding Test

Nominal concentration [mg/L]	Cumulative immobilized Daphnia
	24 hours	48 hours
Control	0	0
0.10	0	0
1.0	0	0
10	10	10
100	10	10

Table 3. Cumulative immobilization data in the Definitive Test

Nominal concentration [mg/L]	Cumulative immobilized Daphnia (Initial Population: 5 per Replicate)
	24 hours						48 hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	5	5	5	5	20	100
3.2	3	3	3	4	13	65	5	5	5	5	20	100
5.6	5	5	5	5	20	100	5	5	5	5	20	100
10	5	5	5	5	20	100	5	5	5	5	20	100

Table 4. Physico-chemical measurements

Nominal concentration [mg/L]		0 hours				24 hours	48 hours
		pH	mg O2/L	%ASV	T [°C]	T [°C]	pH	mg O2/L	%ASV	T [°C]
Control	R1	8.1	9.2	101	20	20	8.1	8.9	100	21
1.0	R1	8.1	9.1	100	20	20	8.1	8.8	99	21
1.8	R1	8.0	9.1	100	20	20	8.1	8.8	99	21
3.2	R1	8.1	9.1	100	20	20	8.1	8.8	99	21
5.6	R1	8.1	9.1	100	20	20	8.1	8.7	98	21
10	R1	8.1	9.1	100	20	20	8.1	8.7	98	21

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the applied guideline were fulfilled.

Conclusions:

The report describes a valid guideline study conducted under certificated GLP compliance. The obtained 48h-EC50 value (1.3 mg/L) leads to a certain toxicity concern of the test substance towards aquatic invertebrates.

Executive summary:

The test substance was investigated for its toxicity towards aquatic invertebrates in accordance to OECD Guideline 202 / EU Method C. 2 (Harris, 2013). Daphnia magna was used as test organism since it is a representative of a wide variety of natural habitats and can therefore be considered as an important non-target organism in freshwater ecosystems. Based on a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test substance at concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours. A control group was maintained under identical conditions but not exposed to the test substance, resulting in 0 % immobilisation. Four replicates per concentration and control group were prepared. Static test conditions were applied and the temperature was held constant at 20 - 21 °C. A photoperiod of 16 h light and 8 h dark cycle with 20 min dawn and dusk transition periods was applied. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 h. Daphnia were considered to be immobilised if there were unable to swim for approx. 15 seconds after gentle agitation. Potassium dichromate served as positive control resulting in a 48h-EC50 of 0.71 mg/L (95 % CL: 0.65 - 0 .76 mg/L). Quantitative analysis of water samples was performed using HPLC/UV (High Performance Liquid Chromatography with UV detection) at 0 and 48 hours. As final result, the EC50(48h) is given as 1.3 mg/L with a 95 % Confidence Limit range of 1.0 - 1.8 mg/L. Throughout the test duration, the preparations were observed to be clear colorless solutions. Validity criteria of the applied guidelines were met.

Description of key information

Daphnia magna_OECD 202/EU Method C.2: EC50(48h): 1.3 mg/L, NOEC(48h): 1.0 mg/L, LOEC(48h): 1.8 mg/L 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

1.3 mg/L

Additional information

Diphenyl methylphosphonate (DPP) was investigated for its toxicity towards aquatic invertebrates in accordance to OECD Guideline 202 / EU Method C. 2 (Harris, 2013). Daphnia magna was used as test organism since it is a representative of a wide variety of natural habitats and can therefore be considered as an important non-target organism in freshwater ecosystems. Based on a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test substance at concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours. A control group was maintained under identical conditions but not exposed to the test substance, resulting in 0 % immobilisation. Four replicates per concentration and control group were prepared. Static test conditions were applied and the temperature was held constant at 20 - 21 °C. A photoperiod of 16 h light and 8 h dark cycle with 20 min dawn and dusk transition periods was applied. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 h. Daphnia were considered to be immobilised if there were unable to swim for approx. 15 seconds after gentle agitation. Potassium dichromate served as positive control resulting in a 48h-EC50 of 0.71 mg/L (95 % CL: 0.65 - 0 .76 mg/L). Quantitative analysis of water samples was performed using HPLC/UV (High Performance Liquid Chromatography with UV detection) at 0 and 48 hours. As final result, the EC50(48h) is given as 1.3 mg/L with a 95 % Confidence Limit range of 1.0 - 1.8 mg/L. Throughout the test duration, the preparations were observed to be clear colorless solutions. Validity criteria of the applied guidelines were met.