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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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: 26/04/11 - Experimental end: 19/05/11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: OECD guideline study performed according GLP criteria.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material was used.
Analytical monitoring:
yes
Details on sampling:
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.
Vehicle:
no
Details on test solutions:
As the test item is soluble in water, stock solutions were prepared the day the test was started.
For the range-finding test a stock solution at 100 mg/L was prepared by weighing 100 mg of test item in 1 liter of dilution water. This stock solution was diluted in order to obtain the convenient range of nominal concentrations: 0.1, 0.5, 1, 5, 10, 50 and 100 mg/L.
For the definitive test, a stock solution at 500 mg/L was prepared by weighing 2 g of test item in 2 liters of dilution water. This stock solution was diluted in order to obtain the convenient range of concentrations: 50, 73, 107, 157, 231, 340 and 500 mg/L.
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) are 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 are 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 104 cells/mL.
The cell density of the pre-culture was about 2.49 x 106 cells/ml for the preliminary test and about 2.39 x 106 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°C +/- 1°C
pH:
See "Any other information on material and methods incl. tables"
Dissolved oxygen:
See "Any other information on material and methods incl. tables"
Salinity:
Test was performed in freshwater.
Nominal and measured concentrations:
For the definitive test, a stock solution at 500 mg/L was prepared by weighing 2 g of test item in 2 liters of dilution water. This stock solution was diluted in order to obtain the convenient range of concentrations: 50, 73, 107, 157, 231, 340 and 500 mg/L.
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.
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. 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:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
310 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 230 - 470
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
200 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 150 - 270
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
73 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
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.

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 31/03/11 were respectively 1.10 mg/L and 0.67 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.

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

2,61E+04

1,21E+05

5,16E+05

52

1,315

500

1,00E+04

1,68E+04

2,68E+04

2,95E+04

3

0,361

340

1,00E+04

2,22E+04

3,34E+04

7,24E+04

7

0,660

231

1,00E+04

2,62E+04

7,61E+04

1,29E+05

13

0,853

157

1,00E+04

2,53E+04

1,04E+05

1,69E+05

17

0,943

107

1,00E+04

2,74E+04

1,31E+05

3,15E+05

31

1,150

73

1,00E+04

3,09E+04

1,50E+05

5,68E+05

57

1,346

50

1,00E+04

2,74E+04

1,19E+05

5,39E+05

54

1,329

The biomass in the control cultures increased exponentially by a factor of 52 (corresponding to a specific growth rate of 1.315 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

500

91,20

72,55

340

82,41

49,81

231

62,66

35,15

157

50,27

28,27

107

23,36

12,56

73

-15,80

-2,41

50

-3,01

-1,10

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

Concentrations ofN,N’- DIETHYL THIOUREA

 

Nominal Concentration

Measured in non inoculated solutions

 

Initial

Final

Final/Initial

(mg/L)

(mg/L)

(mg/L)

%

0

< DL

< DL

-

500

501

502

100.2

340

351

365

104.0

231

257

251

97.7

157

168

174

103.6

107

121

119

98.3

73

83

85

102.4

50

58

58

100

            < DL : concentration lower than the Detection Limit of the analytical method (0.16 mg/L).

           < QL : concentration lower than the Quantification Limit of the analytical method (0.55 mg/L
Validity criteria fulfilled:
yes
Remarks:
See "Overall remarks"
Conclusions:
The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item1,3 – Diethyl-2-thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201.
Algae were exposed to 500 to 50 mg/L 1,3 – Diethyl-2-thiourea dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.
The concentrations of test item causing a 50 % reduction in biomass (EbC50) and in growth rate (ErC50) were estimated respectively at 200 (150-270) and 310 (230-470) 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 1,3 – Diethyl-2-thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to 500 to 50 mg/L 1,3 – Diethyl-2-thiourea dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

The concentrations of test item causing a 50 % reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. It was possible to determine No Observable Effect Concentrations (NOEC). The results were as follows:

 

 

Endpoint

EC50-72h, mg/L (95% CI)

NOEC, mg/L

Cell growth – Biomass (b)

 

200 (150 – 270)

73

Growth rate – Cell multiplication (r)

 

310 (230 – 470)

73

CI: confidence interval

Description of key information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item 1,3 – Diethyl-2-thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201 and GLP requirements. ErC50 was determined: ErC50-72h=310 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
310 mg/L
EC10 or NOEC for freshwater algae:
73 mg/L

Additional information

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 1,3 – Diethyl-2-thiourea for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to 500 to 50 mg/L 1,3 – Diethyl-2-thiourea dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

The concentrations of test item causing a 50 % reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. It was possible to determine No Observable Effect Concentrations (NOEC). The results were as follows:

Endpoint

EC50-72h, mg/L (95% CI)

NOEC, mg/L

Cell growth – Biomass (b)

 

200 (150 – 270)

73

Growth rate – Cell multiplication (r)

 

310 (230 – 470)

73

  CI: confidence intreval