Diphenyl methylphosphonate

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:

2013-04-29 - 2013-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of The United Kingdom

Analytical monitoring:

yes

Details on sampling:

Daily recording was done for water temperature, pH and dissolved oxygen concentrations.
Old and freshly prepared media were investigated at each renewal time for quantitative analysis.

Vehicle:

no

Details on test solutions:

Prior to use the test substance was heated to 50 °C in order to aid weighing. Afterwards the required test substance amount was dissolved directly in dechlorinated tap water with the aid of propeller stirring at approx. 1500 rpm for 24 h and the volumes adjusted to 20 L to give the nominal test concentrations used.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Mean standard length at study initiation: 4.6 cm (SD = 0.2)
- Mean weight at study initiation: 1.21 g (SD = 0.12)
- Feeding during test: none

ACCLIMATION
- Acclimation period: 2013-05-29 - 2013-06-1
- Acclimation conditions: 16 h light, 8 h dark cycle with 20 min dawn and dusk transition periods, 14 - 15 °C water temperature, DOC >= 9.2 mg O2/L (Dissolved Oxygen Concentration)
- Type and amount of food: commercial trout pellets, discontinued approx. 24 h prior to the start of the definitive test
- Health during acclimation: No mortality observed in the 7 days prior to start of the experiment

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period described.

Hardness:

approx. 140 mg/L CaCO3

Test temperature:

14 - 15 °C

pH:

7.9 - 8.4

Dissolved oxygen:

9.8 - 10.0 mg O2/L (95 - 99 % Air Saturation Value; ASV)

Salinity:

Not applicable.

Nominal and measured concentrations:

10, 18, 32, 56 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L glass exposure
- Type: closed
- Aeration: via narrow bore glass tubes
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.42 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Puritie Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dichlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark with 20 min dawn and dusk transition periods

RANGE FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No mortality at test concentrations of 1 and 10 mg/L. Three fish died at 100 mg/L.

Reference substance (positive control):

not required

Remarks:

Not required under the applied guideline.

Duration:

3 h

Dose descriptor:

LC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 18 - 32 mg/L

Duration:

6 h

Dose descriptor:

LC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 18 - 32 mg/L

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 19 - 26 mg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 19 - 26 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 19 - 26 mg/L

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 17 - 25 mg/L

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- The test preparations were observed to be clear colorless solutions throughout the 96 h exposure period.
-Sublethal effects were observed at test concentrations of 18 mg/L and above. These responses were loss of equilibrium, twitching, swimming at bottom of tank and swollen abdomen.
- After approx. 1 h exposure all fish at 32 mg/L were observed to exhibit prolonged sub-lethal effects. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.
- The highest test concentration where no sub-lethal effects were observed during the exposure period of 96 h was 10 mg/L.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Trimmed Spearman-Karber method (Hamilton et al., 1977) using the ToxCalc computer software package (ToxCalc, 1999).

Sublethal observations / clinical signs:

Table 2. Cumulative mortality data in the Range-finding experiment

Nominal concentration [mg/L]	Cumulative Mortality (Initial population: 3)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
1.0	0	0	0	0	0	0
10	0	0	0	0	0	0
100	3	3	3	3	3	3

Table 3. Cumulative mortality data in the definitive experiment

Nominal concentration [mg/L]	Cumulative Mortality (Initial population: 7)						% Mortality
	3h	6h	24h	48h	72h	96h	96h
Control	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0
18	0	0	1	1	1	2	29
32	7	7	7	7	7	7	100
56	7	7	7	7	7	7	100
100	7	7	7	7	7	7	100

 

Table 4. Sub-lethal effects of exposure in the definitive experiment

Nominal concentration [mg/L]	Sub-lethal effects	Time (hours)
		3	6	24	48	72	96
Control	No abnormalities detected.	7/7	7/7	7/7	7/7	7/7	7/7
10	No abnormalities detected.	7/7	7/7	7/7	7/7	7/7	7/7
18	No abnormalities detected.	7/7	7/7	6/6
	Swimming at bottom of tank				6/6	6/6	5/6
	Swollen abdomen and loss of equilibrium						1/6**
32	Loss of equilibrium and twitching	7/7*	A/D
56	No abnormalities detected.	A/D
100	No abnormalities detected.	A/D

* After approximately 1 hour exposure all fish at the 32 mg/L were observed with a loss of equilibrium and twitching. Due to the approach of the substantial severity limit these fish were killed and classed as mortalities for the 3-h time point.

** After approximately 94 hours exposure 1 of the remaining 6 fish at the 18 mg/L was observed with a swollen abdomen and loss of equilibrium. Due to the approach of the substantial severity limit these fish were killed and classed as mortalities for the 96-h time point.

A/D = All fish dead

Table 5. Physico-chemical measurements

Nominal concentration [mg/L]	Time (hours)
	0 h (fresh media)				24 h (old media)				24 h (fresh media)				48 h (old media)
	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C
Control	8.1	10.0	99	15	8.4	10.0	97	14	8.0	10.0	99	15	8.4	10.0	99	15
10	8.1	9.9	96	14	8.4	9.9	96	14	8.0	10.0	99	15	8.4	10.0	97	14
18	8.1	9.9	96	14	8.4	10.0	97	14	8.0	9.9	98	15	8.3	9.9	96	14
32	8.0	9.9	96	14	8.0	10.0	97	14		A/D				A/D
56	8.0	10.0	97	14	8.0	10.0	97	14		A/D				A/D
100	8.0	10.0	97	14	8.0	10.0	97	14		A/D				A/D

 

Nominal concentration [mg/L]	Time (hours)
	48 h (fresh media)				72 h (old media)				72 h (fresh media)				96 h (old media)
	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C	pH	mg O2/L	% ASV	°C
Control	7.9	9.7	96	15	8.2	9.6	93	14	7.9	9.8	95	14	8.3	10.2	99	14
10	7.9	9.7	96	15	8.3	9.9	96	14	8.0	9.8	95	14	8.3	10.2	99	14
18	7.9	9.7	96	15	8.3	9.8	95	14	8.0	9.8	95	14	8.3	10.2	99	14
32		A/D				A/D				A/D				A/D
56		A/D				A/D				A/D				A/D
100		A/D				A/D				A/D				A/D

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of applied guideline are fulfilled.

Conclusions:

The report describes a valid guideline study conducted under certificated GLP compliance. The obtained 96h-EC50 value (20 mg/L) leads to a certain toxicity concern of the test substance towards fish.

Executive summary:

The acute toxicity of Diphenyl methylphosphonate (DPP) was assessed according to OECD Guideline 203 / EU Method C.1 (Harris, 2013). Based on a preliminary range-finding investigation, Oncorhynchus mykiss was exposed in groups of seven to an aqueous solution of the test substance over a concentration range of 10, 18, 32, 56 and 100 mg/L. The exposure period was set as 96 hours at a temperature of 14 - 15 °C under semi-static test conditions. A daily renewal of the test preparation was employed to ensure that the concentrations of the test substance remained near nominal and to prevent the build up of nitrogenous waste products. Mortalities and sub-lethal effects in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation. Water samples were taken from the control and all surviving test groups, from fresh test solutions at 0 and 72 h and aged solutions at 24 and 96 h for quantitative analysis. Physico-chemical parameters (i.e. pH, Dissolved Oxygen Concentrations (DOC), temperature) were recorded daily. Twitching, loss of equilibrium, swollen abdomen and swimming at the bottom of the tank were observed as sub-lethal effects at test concentrations of 18 mg/L and above. The highest concentration without sub-lethal effects and mortality was 10 mg/L. At 32 mg/L all fish were dead. Throughout the whole exposure period all test preparations were observed to be clear colorless solutions. As final result, the LC50(96h) is given as 20 mg/L with a 95 % Confidence Limit of 17 - 25 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 18 mg/L. The No Observed Effect Concentration (NOEC) was 10 mg/L. All test concentrations are given as nominal results. The validity criteria given in the applied guideline were fulfilled.

Description of key information

Oncorhynchus mykiss_OECD 203/EU Method C.1: LC50(96h): 20 mg/L, LOEC(96h): 18 mg/L, NOEC(96h): 10 mg/L 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

20 mg/L

Additional information

The acute toxicity of Diphenyl methylphosphonate (DPP) was assessed according to OECD Guideline 203 / EU Method C.1 (Harris, 2013). Based on a preliminary range-finding investigation,Oncorhynchus mykiss was exposed in groups of seven to an aqueous solution of the test substance over a concentration range of 10, 18, 32, 56 and 100 mg/L. The exposure period was set as 96 hours at a temperature of 14 - 15 °C under semi-static test conditions. A daily renewal of the test preparation was employed to ensure that the concentrations of the test substance remained near nominal and to prevent the build up of nitrogenous waste products. Mortalities and sub-lethal effects in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation. Water samples were taken from the control and all surviving test groups, from fresh test solutions at 0 and 72 h and aged solutions at 24 and 96 h for quantitative analysis. Physico-chemical parameters (i.e. pH, Dissolved Oxygen Concentrations (DOC), temperature) were recorded daily. Twitching, loss of equilibrium, swollen abdomen and swimming at the bottom of the tank were observed as sub-lethal effects at test concentrations of 18 mg/L and above. The highest concentration without sub-lethal effects and mortality was 10 mg/L. At 32 mg/L all fish were dead. Throughout the whole exposure period all test preparations were observed to be clear colorless solutions. As final result, the LC50(96h) is given as 20 mg/L with a 95 % Confidence Limit of 17 - 25 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 18 mg/L. The No Observed Effect Concentration (NOEC) was 10 mg/L. All test concentrations are given as nominal results. The validity criteria given in the applied guideline were fulfilled.

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