1,3-diphenyl-2-thiourea

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 16/05/11 - Experimental end: 14/06/11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

After 24, 48, and 72 h of incubation, a volume of 200 µL was sampled from each test flask, pipetted into a quartz microplate.

Vehicle:

no

Details on test solutions:

For the range-finding test a stock solution was prepared by weighing 100 mg of test item in 1 liter of dilution water. After 72 h under stirring, the mixture was filtered on a Millipore membrane HAWP04700 at 0.45 µm.
This saturated stock solution was then diluted in order to obtain the convenient range of nominal concentrations: 0.1, 0.5, 1, 5, 10, 50 and 100% of saturation.
For the definitive test, a stock solution was prepared by weighing 100 mg of test item in 1 liter of dilution water. After 90 h under stirring, the mixture was filtered on a Millipore membrane HAWP04700 at 0.45 µm. This stock solution was diluted in order to obtain the convenient range of nominal concentrations: 5,8.2, 13.5, 22.2, 36.8, 60.6 and 100% of saturation.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) were obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).
Two flasks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken.
These stock cultures were renewed every week, using two new cultures.
The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use.
Three days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged.
At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to get an initial cell concentration of 10^4 cells/mL.
The cell density of the pre-culture was about 2.49 x 10^6 cells/ml for the preliminary test and about 2.39 x 10^6 cells/ml for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post-exposure observation period was done.

Test temperature:

23 ± 1°C

pH:

See "Any other information on Materials and Methods including tables".

Dissolved oxygen:

See "Any other information on Materials and Methods including tables".

Salinity:

Test was performed in freshwater.

Nominal and measured concentrations:

Based on the results of the preliminary test, the 7 nominal concentrations tested were: 5 ; 8.2 ; 13.5 ; 22.2 ; 36.8 ; 60.6 and 100% of saturation. Practically, the initial measured concentrations were: 1.7 ; 2.8 ; 4.7 ; 7.7 ; 12.7 ; 20.9 ; 34.7 mg/L. Initial measured concentrations remained very stable during the study.

Details on test conditions:

The incubation was performed in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C; flasks were continuously shaken with a rotation at 20 rpm and constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33). The study was performed using 120 mL glass bottles stoppered with cellulose bungs. Filling volume: 50 mL.

All concentrations were prepared as triplicates. Flasks were stoppered with cellulose bungs and placed in a phytoculture cabinet (Strader DCS Pulsar).
An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. This was used for determining algae growth. A non-inoculated blank (labelled Bl) containing only dilution water without test item was also prepared and incubated.
Test flasks and blanks were prepared in triplicate. Six replicates were used for the control.
After 24, 48 and 72 h of incubation, about 1 mL was sampled from each test flask. After 24 and 48 hours test flasks were replaced in the same position in the rotary shaker. Samples were stored in darkness until determination of algae concentration by cell counter cell as. Measured concentrations of algae were used for calculating the percentages inhibition and plotting the growth curves.
The pH and dissolved O2 were measured for all concentrations, at the beginning and the end of the test.

Reference substance (positive control):

yes

Remarks:

See "Any other information on material and methods incl. tables"

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Analysed with Toxrat 3.2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Analysed with Toxrat 3.2

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. Microscopic observation confirmed that the algae appeared normal at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm. EC50 and EC10 for growth rate inhibition were 4.7 and 14 mg/L respectively.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The nearest values of ErC50 and EbC50 obtained on 17/06/11 were respectively 0.90 mg/L and 0.48 mg/L.
For information, ISO 8692 reports the following results for an inter-laboratory exercise on potassium dichromate: ErC50: 0.60 to 1.03 mg/L EbC50: 0.20 to 0.75 mg/L.

Reported statistics and error estimates:

Results were analysed by the study director with an Excel sheet. EC50 was determined graphically and no EC10 was determined. The registrant re-analysed statistically the raw data of the study with Toxrat 3.2.0 to obtain good quality ECx determination.

Average cell densities, biomass (A) and growth rates (µ)

Concentration (nominal)

Average cell density (cell/mL)

mg/L

T0

T24h

T48h

T72h

A

µ

0 (T)

1,00E+04

3,81E+04

1,76E+05

7,92E+05

79

1,457

100

1,00E+04

1 ,33E+04

2,22E+04

1,98E+04

2

0,227

60.6

1,00E+04

2,34E+04

3,61E+04

3,56E+04

4

0,423

36.8

1,00E+04

2,64E+04

5,62E+04

1,41E+05

14

0,882

22.2

1,00E+04

2,74E+04

7,87E+04

1,94E+05

19

0,989

13.5

1,00E+04

2,97E+04

1,69E+05

6,49E+05

65

1,391

8.2

1,00E+04

3,05E+04

1,75E+05

7,40E+05

74

1,435

5

1,00E+04

3,30E+04

1,68E+05

7,10E+05

71

1,421

 

In addition, Annex 2 presents ratios (R) of cell densities and growth factors (k) for each test concentration and for the control.The biomass in the control cultures increased exponentially by a factor of 79 (corresponding to a specific growth rate of 1.457 day^-1) within the 72-hour test period,which is higher than the minimal value (R = 16) mentioned OECD guideline 201.

Definitive test - Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

Concentration (nominal)	IAi	Iµi
(mg/L)	(%)	(%)
0 (T)	0,00	0,00
100	96,51	84,43
60.6	91,06	70,98
36.8	78,13	39,48
22.2	69,53	32,13
13.5	14,95	4,55
8.2	5,87	1,53
5	9,27	2,49

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

Concentrations ofN,N’- DIPHENYL THIOUREA
Nominal Concentration	Measured in non-inoculated solutions
	Initial	Final	Final/Initial
(mg/L)	(mg/L)	(mg/L)	%
0	< DL	< DL	-
5	1.70	1.70	100
8.2	2.80	2.80	100
13.5	4.66	4.76	102.1
22.2	7.65	7.60	99.3
36.8	12.7	12.9	101.6
60.6	20.9	21.2	101.4
100	34.7	34.9	100.6

 

 

           < DL : concentration lower than the Detection Limit of the analytical method (0.0088 mg/L).

           < QL : concentration lower than the Quantification Limit of the analytical method (0.029 mg/L).

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks"

Conclusions:

The determination of the growth rate inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item N,N’- Diphenyl Thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201. EC50 was 14 mg/L and EC10 was 4.7 mg/L.

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item N,N’- Diphenyl Thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to graded series of N,N’- Diphenyl Thiourea concentrations. Growth rate inhibition was estimated over a 3 -day period. The concentrations of test item causing 50% and 10% reduction in growth rate (ErCx) were estimated by the registrant from the raw data included in the study report with Toxrat 3.2.0. (see attached file). EC50 was 14 mg/L and EC10 was 4.7 mg/L for growth rate inhibition.

Description of key information

The toxicity was assessed according to the OECD Guideline 201.
 

72h-ErC50 = 14 mg/L
72h-ErC10 = 4.7 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

14 mg/L

EC10 or NOEC for freshwater algae:

4.7 mg/L

Additional information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item N,N’- Diphenyl Thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to graded series of N,N’- Diphenyl Thiourea concentrations. Growth rate inhibition was estimated over a 3-day period. The concentrations of test item causing 50% and 10% reduction in growth rate (ErCx) were estimated by the registrant from the raw data included in the study report with Toxrat 3.2.0. EC50 was 14 mg/L and EC10 was 4.7 mg/L for growth rate inhibition.