Bis(piperidinothiocarbonyl) hexasulphide

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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

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 DIPENTAMETHYLENETHIURAM HEXASULFIDE in 1 liter of dilution water during 1 h under ultrasonic treatment. 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):

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).

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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

23 ± 1°C

pH:

see "Any other information on results incl. tables"

Dissolved oxygen:

see "Any other information on results incl. tables"

Salinity:

Not applicable

Nominal and measured concentrations:

Preliminary test, nominal concentrations: 100, 50, 10, 5, 1, 0.5, 0.1 mg/L
Definitive test (limit test), nominal concentration: 100 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.

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).

Preliminary test: 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.

Definitive test: number of replicates: 3 (6 for the control). 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.
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.
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:

NOEC

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 7.9 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 18/10/12 were respectively 1.10 mg/L and 0.64 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.

Validity criteria fulfilled:

yes

Conclusions:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item DIPENTAMETHYLENETHIURAM HEXASULFIDE 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 DIPENTAMETHYLENETHIURAM HEXASULFIDE dissolved in dilution water (limit test). DIPENTAMETHYLENETHIURAM HEXASULFIDE EC50-72h values were determined to be higher than the solubility limit of the test item in the conditions of the test.

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item DIPENTAMETHYLENETHIURAM HEXASULFIDE 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 DIPENTAMETHYLENETHIURAM HEXASULFIDE 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 growth rate (ErC50) were estimated. The results were as follows (values based on geometric average value of initial and final measured concentrations):

 

ErC50-72h: > 7.9 µg/L (100 mg/L, nominal concentration)

 

This value is the solubility limit of the test item in the conditions of the test. DIPENTAMETHYLENETHIURAM HEXASULFIDE EC50-72h values are thus higher than the solubility limit of the test item.

Additional information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item DIPENTAMETHYLENETHIURAM HEXASULFIDE 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 DIPENTAMETHYLENETHIURAM HEXASULFIDE 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 growth rate (ErC50) were estimated. The results were as follows (values based on geometric average value of initial and final measured concentrations):

 

ErC50-72h: > 7.9 µg/L (100 mg/L, nominal concentration)

 

This value is the solubility limit of the test item in the conditions of the test. DIPENTAMETHYLENETHIURAM HEXASULFIDE EC50-72h values are thus higher than the solubility limit of the test item.