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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2011- October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
COPPER DIBUTYLDITHIO CARBAMATE was measured in non-inoculated test solutions (without algae) at the beginning and at the end of the test at the highest concentration tested (100 mg/L).

Storage of samples: None
Samples treatment: Dilution with methanol
Vehicle:
no
Details on test solutions:
For the range finding test and for the definitive test a stock parent solution at 100 mg/L of the test item was prepared three days before the starting test by mixing 100 mg of the test item COPPERDIDUTYLTHIO CARBAMATE in 1 liter of dilution water during 66 h. This solution was filtered through an ester cellulose Millipore filter 0.45µm (HAWP 04700) to obtain limpid solution. It was used to prepare the convenient range of nominal concentrations: 100, 50, 10, 5, 1, 0.5 and 0.1 mg/L. For the definitive test, the same procedure was used to prepare a 100 mg/L solution as a WAF (limit test).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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). The conditions used for culturing algae are described in Annex 2 of the OECD 201 guideline. 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 10^4 cells/mL. The cell density of the pre-culture was about 1.82 x 10^6 cells/ml for the preliminary test and about 1.39 x 10^6 cells/ml for the definitive test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
No data
pH:
from 7.98 to 10.8
Dissolved oxygen:
from 9.2 to 10.4 mg/L
Salinity:
Water media is a freshwater.
Nominal and measured concentrations:
Preliminary test (nominal concentrations): 100, 50, 10, 5, 1, 0.5 and 0.1 mg/L.
Definitive test: 100 mg/L (nominal concentration), 0.66 µg/L (initial measured value)
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. 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. Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device in the range-finding test. In the definitive test, cells were numbered with a cell counter (Beckman Coulter Z2).

In the preliminary test, algae were exposed under static conditions to a series of 7 concentrations (100, 50, 10, 5, 1, 0.5, 0.1 mg/L) in dilution water (composition detailed in Annex 1). The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. All concentrations were prepared as duplicates. 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 and test item was also prepared and incubated. After 24, 48, and 72 h of incubation, a volume of 200 µL was sampled from each test flask, pipetted into a quartz microplate. Fluorescence was then determined using a cytofluorimeter and by comparison with a calibration range, cell density was determined, according to the measured fluorescence. Dissolved O2 and pH were measured in the control and the highest concentration, in non-inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.

Based on the results of preliminary test and as results of the preliminary test showed no toxicity even at the higher nominal concentration, it was decided to run a limit test at 100 mg/L (nominal concentration). Number of replicates: 3 (6 for the control). 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 under. 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.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.207 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
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 June 01 2012 were respectively 0.75 mg/L and 0.54 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.

Definitive test

 

Concentrations (nominal) mg/L

pH

Dissolved O2 (mg/L)

T0

T72h

T0

T72h

0 (Bl)

7.98

7.97

9.2

8.7

0 (T)

-

9.75

-

10.1

100 (a)

7.99

10.08

9.5

10.4

An increase in the pH is observed. This may be associated to consumption of the dissolved CO2 due to the growth of algae.

Definitive test

Concentration

(nominal)

mg/L

Average cell density (cell/mL)

A

µ

T0

T24h

T48h

T72h

0 (T)

1.00E+04

5.26E+04

1.90E+05

9.47E+05

95

1.517

100 (a)

1.00E+04

4.44E+04

1.79E+05

9.47E+05

95

1.517

Validity criteria fulfilled:
yes
Remarks:
see conclusions
Conclusions:
- The biomass in the control cultures has increased exponentially by a factor of 95 higher than 16 within the 72-hour test period. This corresponds to a specif c growth rate of 0.92 day-1.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.
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 COPPER DIBUTYLDITHIO CARBAMATE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to one concentration of 100 mg/L of COPPER DIBUTYLDITHIO CARBAMATE dissolved in dilution water (limit test). 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. The results were as follows (EC50-72h values based on geometric average value of initial and final measured concentrations):

 

EC50-72h> 0.207µg/L (biomass and growth rate)

Description of key information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item COPPER DIBUTYLDITHIO CARBAMATE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to one concentration of 100 mg/L of COPPER DIBUTYLDITHIO CARBAMATE dissolved in dilution water (limit test). The results were as follows (EC50-72h values based on geometric average value of initial and final measured concentrations): 
 
EC50-72h> 0.207µg/L (biomass and growth rate), i.e. no effect was observed up to the limit of solubility.

Key value for chemical safety assessment

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 COPPER DIBUTYLDITHIO CARBAMATE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to one concentration of 100 mg/L of COPPER DIBUTYLDITHIO CARBAMATE dissolved in dilution water (limit test). 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. The results were as follows (EC50-72h values based on geometric average value of initial and final measured concentrations):

 

EC50-72h> 0.207µg/L (biomass and growth rate)