1,3-diphenyl-2-thiourea

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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: None.
Samples treatment: Dilution with acetonitrile.

Vehicle:

no

Details on test solutions:

The saturated stock solution of N,N’- DIPHENYL THIOUREA was obtained by mixing of 100 mg of the test item into 1 L of dilution water, under 3 hours of magnetic stirring, followed by a 0.45 µm filtration (Millipore HAWP04500). The filtrated stock solution was clear and colourless.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The organisms used for the test were Danio rerio (Teleostei, Cyprinidae). The batch n°11/Br/03/1 was supplied by Elevage de la Grande Rivière France (69490 Saint Forgeux). The fish selected for the test were all from the same batch and were of the same age (date of birth stated by the supplier: 28/06/11). Their size (determined by measuring 10 randomly sampled fish) was 35 ± 4 mm. The mortality in the aquarium containing the batch of fish used for the tests was less than 5 % (observed mortality: 0.7%) over the 7 days preceding the test. The sensitivity of the biological reagent is checked at least once for each new batch of fish, by determining the LC50-24h of the potassium dichromate. For reference, the results of the last test, carried out on 18/10/2011, were a LC50-24h of 220 mg/L (potassium dichromate Merck 1.02403.0080 batch n°102403H). For reference, the former AFNOR standard NF T 90-303 states that the LC50-24h of the potassium dichromate with respect to Danio rerio must be between 200 and 400 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Not specified

Test temperature:

The temperature, which was measured on a continuous basis in a test flask, was maintained between 21 and 22 °C

pH:

See tables below

Dissolved oxygen:

See tables below

Salinity:

Not applicable

Nominal and measured concentrations:

Preliminary test: 100, 50, 10, 5, 1 % of saturated stock solution of N,N’- DIPHENYL THIOUREA in dilution water.
Definitive test, nominal concentrations tested: 100, 56.2, 31.6, 17.8 and 10 % of saturated stock solution of N,N’- DIPHENYL THIOUREA (factor 1.78).

See table below for details

Details on test conditions:

The room was equipped with a programmed lighting system which produced a controlled lighting sequence: intensity between 400 and 800 lux at the surface of the aquariums; photo-period of 12 hours of darkness per 24 h.
The study was performed using aquaria with a nominal capacity of 5 litres, covered with Altuglas® cover plates drilled with holes to allow the test solutions to be aerated. Volume of test medium per aquarium was 2 x 2.5 L.
Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement.

Preliminary test: series of 5 concentrations: 100, 50, 10, 5, 1 % of saturated stock solution of N,N’- DIPHENYL THIOUREA in dilution water.
Five fish were added per aquarium, only one vessel was prepared for each concentration.
The pH and the dissolved O2 were measured at the beginning and at the end of the test, at the highest concentration and in the control vessel.

Definitive test, nominal concentrations tested: 100, 56.2, 31.6, 17.8 and 10 % of saturated stock solution of N,N’- DIPHENYL THIOUREA (factor 1.78).
At the beginning of the test, appropriate volumes of the stock solution of N,N’- DIPHENYL THIOUREA were poured directly into each test aquarium, which was then topped up with dilution water to obtain the above mentioned range of nominal concentrations (no pH-adjustment done).
Number of fish: 10 for each test concentration and for the control.
The definitive test was carried out under static conditions.
Every day, the dissolved oxygen and the pH were measured at each concentration and in the control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 33 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No 96 -h LC50 could be determined because fish mortality was lower than 50% up to the limit of DPTU solubility in water.

Results with reference substance (positive control):

The sensitivity of the biological reagent is checked at least once for each new batch of fish, by determining the LC50-24h of the potassium dichromate. For reference, the results of the last test, carried out on 18/10/2011, were a LC50-24h of 220 mg/L (potassium dichromate Merck 1.02403.0080 batch n°102403H). For reference, the former AFNOR standard NF T 90-303 states that the LC50-24h of the potassium dichromate with respect to Danio rerio must be between 200 and 400 mg/L.

Reported statistics and error estimates:

The mortality data are analyzed using an Excel sheet program. It was designed to calculate the LC50 value and the 95% confidence interval. Probit analysis is generally used to calculate the 24-, 48-, 72- and 96-hour LC50 values.

Sublethal observations / clinical signs:

Measured pH in the different test systems

Stock solution dilution (%)	T0	T24h	T48h	T72h	T96h
100	7.69-7.67	7.39-7.33	7.44-7.55	7.52-7.59	7.65-7.62
56.2	7.66-7.65	7.30-7.42	7.43-7.55	7.46-7.54	7.51-7.52
31.6	7.39-7.37	7.36-7.36	7.51-7.51	7.54-7.53	7.62-7.55
17.8	7.19-7.21	7.38-7.32	7.54-7.52	7.57-7.54	7.63-7.51
10	7.12-7.12	7.27-7.26	7.47-7.45	7.55-7.47	7.67-7.48
0 (control)	7.08-7.09	7.73-7.51	7.80-7.60	7.77-7.64	7.87-7.65

 

 

Measured O2 concentrations (% of saturation) in the different test systems

 

Stock solution dilution (%)	T0	T24h	T48h	T72h	T96h
100	97-96	73-70	76-85	80-84	86-84
56.2	96-96	70-76	76-84	75-81	78-78
31.6	97-96	76-76	82-81	82-80	85-81
17.8	97-96	75-69	83-80	83-81	87-78
10	97-96	66-66	77-76	82-75	88-75
0 (control)	97-97	88-76	93-85	91-84	96-85

 

 

Nominal, measured and effective concentrations of the test item at the beginning and at the end of the exposure period

 

Stock solution dilution (%)	Measured concentrations of N,N’- DIPHENYL THIOUREA
	Initial (mg/L)	Final (mg/L)	Final/Initial (%)	Effective concentrations*
100	33.3	34.4	103	33.3
56.2	18.8	19.5	104	18.8
31.6	10.6	10.6	100	10.6
17.8	5.98	5.51	92	5.98
10	3.30	2.81	85	3.30

 

< DL : concentration lower than the Detection Limit of the analytical method (0.044 mg/L).

< QL : concentration lower than the Quantification Limit of the analytical method (0.15 mg/L).

 

* According to OECD guidance document on aquatic toxicity testing of difficult substances and mixtures (n°23):

- For static and semi-static and flow-through tests, where the concentrations remain within 80-120% of nominal, the effect concentrations can be expressed relative to nominal or measured concentrations;

- For static and semi-static tests, where the concentrations do not remain within 80-120% of nominal, the effect concentrations could be determined and expressed relative to the geometric mean of the measured concentrations. As the final concentrations remain within 80-120 % of the initial concentrations, the initial concentrations were used as effective concentrations for the calculation.

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Conclusions:

The study was performed in compliance with the following quality criteria:
· The mortality in the control did not exceed 10 per cent at the end of the test;
· The concentration of dissolved oxygen in the test vessels remained above 60 % of the air saturation value at the end of the test;
· The pH did not vary by more than 1 unit.

Executive summary:

The acute toxicity (assessed by lethality) of the test item N,N’- DIPHENYL THIOUREA to Danio rerio (freshwater fish) for a duration of 96 hours was assessed according to the OECD Guideline 203. Fish were exposed under static conditions to a range of concentrations of N,N’- DIPHENYL THIOUREA dissolved in dilution water. No 96 -h LC50 could be determined because fish mortality was lower than 50% up to the limit of DPTU solubility in water:

LC50 -96h > 33 mg/L

Description of key information

The acute toxicity assessed according to the OECD Guideline 203.
96h-LC50 > limit of solubility of N,N’- DIPHENYL THIOUREA in water

Additional information

The acute toxicity (assessed by lethality) of the test item N,N’- DIPHENYL THIOUREA to Danio rerio (freshwater fish) for a duration of 96 hours was assessed according to the OECD Guideline 203. Fish were exposed under static conditions to a range of concentrations of N,N’- DIPHENYL THIOUREA dissolved in dilution water. No 96h- LC50 could be determined because fish mortality was lower than 50% up to the limit of solubility of N,N’- DIPHENYL THIOUREA in water:

 

96h-LC50 > limit of solubility of N,N’- DIPHENYL THIOUREA in water