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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Storage of samples: No
Samples treatment: Dilution with acetonitrile
Details on test solutions:
For the range finding test and for the definitive test, a stock solution at 100 mg/L of the test item was prepared the day of the starting test by mixing 100 mg of the test item N,N’-DIPHENYL THIOUREA in 1 liter of dilution water. This solution was stirred during 5 h and was filtered through ester cellulose Millipore filter 0.45µm (HAWP 04700) to obtain limpid solution.

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna Straus (Cladocera, Crustacea), clone 5 or clone A. Animals come from a stock breeding in the laboratory reared in VOLVIC® water added with vitamin B12, Na2SeO3, 5H2O, calcium and magnesium salts and fed with unicellular green fresh-water algae Pseudokirchneriella subcapitata and Chlorella Vulgaris. Selection of test organisms less than 24 hours-old is made by filtration.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Not specified
Test temperature:
The temperature, continuously recorded in one test flask, remained between 20.1 and 20.6°C and was constant w ithin ± 1°C
pH:
see "Any other information on results incl. tables"
Dissolved oxygen:
see "Any other information on results incl. tables"
Salinity:
NC
Nominal and measured concentrations:
Preliminary test, range of nominal concentrations: 100, 50, 10, 5, 1 and 0.1 mg/L.
Definitive test, range of nominal concentrations: 100, 54.8, 30.0, 16.8, 9.2 and 5 mg/L.
Details on test conditions:
The study was performed in a temperature-controlled room using 30 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps. The temperature, continuously recorded in one test flask, remained between 20.1 and 20.6°C and was constant w ithin ± 1°C according to OECD 202 Guideline.

Physical-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.

Water: ultra-pure water was used for the preparation of dilution water (resistivity > 18M).

Definitive and preliminary test: approximately 90% of the final volume of the test solution was added into test flasks. Five Daphnia magna aged from 6 to 24 hours were added into each test flask, which were then totally filled with the test solution (30 mL)

In the preliminary test, daphnids were exposed under static conditions to a series of 6 concentrations (100, 50, 10, 5, 1, 0.1 mg/L) of N,N’-DIPHENYL THIOUREA in dilution water.. Two replicates were prepared for each concentration. Two control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature of 20 °C.

After 48 hours of incubation, mobile Daphnia magna were counted.

Dissolved O2 and pH were measured at the highest tested concentration and in the control.


Based on the results of preliminary test, the concentrations tested were: 100, 54.8, 30, 16.8, 9.2 and 5 mg/L. Volumetric flasks were filled with the required volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 100 mg/L to 5 mg/L.

The definitive test was performed with a concentration range forming a geometric progression with a factor of 1.82. Four replicates were prepared for each concentration. Four control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature of 20.3 ± 0.3°C.

Dissolved O2 and pH were measured at the highest concentration and in the control at the beginning and at all concentration and in the control the end of the test
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL (1.7-3.5)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
10.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL (7.9-15)
Details on results:
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.
EC10 and EC50 calculated by Probit analysis have been recalculated using the software ToxRat.
Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated every month by performing an inhibition test on potassium dichromate. The nearest value of EC50 obtained on May 2012 was 0.76 mg/L For reference, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.
Reported statistics and error estimates:
Originally, the inhibition of mobility data were analyzed using an Excel program. It was designed to calculate the EC50 and the EC10 values and the 95% confidence intervals.
EC10 and EC50 have been recalculated using the software ToxRat (v.3.3.0)

Definitive test - Percentage of Daphnia magna immobilisation

















































Concentration (extrapolated)


mg/L



Immobilisation (%)



24 h



48 h



32.2



0



80



15.3



0



65



8.9



0



50



5.0



0



25



2.5



0



15



1.1



0



0



< DL



0



0



 


Dissolved O2 and pH measured at the beginning and at the end (48h) of the exposition




























































Nominal concentration mg/L



pH



Dissolved O2 (mg/L)



T0



T48h



T0



T48h



0 (T)



8.12



7.66



9.0



8.4



5



7.96



7.70



8.3



9.2



7.96



7.77



8.4



16.8



7.99



7.76



8.4



30



7.98



7.77



8.4



54.8



7.98



7.78



8.5



100



7.99



7.85



8.6



 


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

































































Nominal


(mg/L)



Measured in solutions



Extrapolated* Initial (mg/l)



Initial


(mg/l)



Final


(mg/l)



Final/Initial


%



100



33.6



30.9



92



32.2



54.8



17.8



13.1



73



15.3



30



9.57



8.35



87



8.9



16.8



5.57



4.55



82



5.0



9.2



3.02



2.12



70



2.5



5



1.71



0.72



42



1.1



0



< DL



< DL



-



< DL



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


< DL: concentration lower than the Quantification Limit of the analytical method (0.045mg/L).


 


* Concentrations calculated using geometric mean


 


According to OECD guidance document on aquatic toxicity testing of difficult substances and mixtures


(n°23):


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


 


- For 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 ratio is lower than 80% for some final concentrations, extrapolated concentrations are used for the calculation of effective concentrations


 

Validity criteria fulfilled:
yes
Remarks:
see conclusions
Conclusions:
The acute toxicity (inhibition of mobility) of the test item N,N’-DIPHENYL THIOUREA for a duration of 48 hours was assessed according to the OECD Guideline 202 under GLP.
 
The study was performed in compliance with the following quality criteria:

· The immobilisation in the control did not exceed 10 per cent at the end of the test;
· Daphnids in the control were not trapped at the surface of the water;
· The dissolved oxygen concentration remained above 3 mg/L over the test period.
Executive summary:

The acute toxicity (inhibition of mobility) of the test item N,N’-DIPHENYL THIOUREA for a duration of 48 hours was assessed according to the OECD Guideline 202 under GLP. Daphnia magna were exposed under static conditions to a range of concentrations of N,N’-DIPHENYL THIOUREA dissolved in dilution water. The toxic effect measured during the assay was the inhibition of mobility over time periods of 24 and 48 hours.


 


The concentration of test item causing a 50% reduction in mobility (EC50) was estimated as follows (geometric mean of measured concentration):


 


EC50-48h = 10.2 mg/L


 

Description of key information

The acute toxicity was assessed according to the OECD Guideline 202.

 
48h-EC50 = 10.2 mg/L
 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10.2 mg/L

Additional information

The acute toxicity (inhibition of mobility) of the test item N,N’-DIPHENYL THIOUREA for a duration of 48 hours was assessed according to the OECD Guideline 202 under GLP. Daphnia magna were exposed under static conditions to a range of concentrations of N,N’-DIPHENYL THIOUREA dissolved in dilution water. The toxic effect measured during the assay was the inhibition of mobility over time periods of 24 and 48 hours.


 


The concentration of test item causing a 50% reduction in mobility (EC50) was estimated as follows (geometric mean of measured concentration):


 


48h-EC50 = 10.2 mg/L