Disulfiram

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Oct 2019 - 10 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test item concentration and conntrol
- Sampling method: At the start of the test (0 hours), 8 mL samples of freshly prepared test media were taken from the control and the test media preparation vessels for chemical analysis. At 24, 48 and 72 hours, 8 mL samples were also taken for chemical analysis from the pooled old test media from the test and control groups. In each case triplicate samples were taken, one for chemical analysis and two as ‘back-up’ samples should further analysis be required.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, amounts (0.63, 1.29, 2.47, 5.09 and 10.07 mg) of test substance were separately added to 1000 mL of OECD medium. The preparations were stirred for ca 24 hours followed by a ca 1 hour settlement period after which the aqueous phase of each preparation was removed.
- Controls: The control was prepared by adding OECD medium only to the vessels.
- Test concentration separation factor: 2.0
- Evidence of undissolved material: The freshly prepared (new) test media for the control and test concentrations at the start of the test were observed to be colourless solutions.After 72 hours the test solution appeared as a homogenous, hazy dispersion of algae cells

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Raphidocelis subcapitata (Strain 278/4; formerly known as Pseudokirchneriella subcapitata)
- Source: originally obtained from the Culture Collection of Algae and Protozoa (CCAP), Oban, UK
- Age of inoculum (at test initiation): Culture was incubated for ca 72 hours prior to the start of each test.
- Method of cultivation: Prior to the definitive test, two starter cultures were prepared from a current master culture at an inoculum concentration of 1 x 10E^04 cells/mL (1 mL of the master culture added to 100 mL of OECD media); incubation conditions were the same as used in the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 – 22.4 °C

pH:

Control: 7.82 - 9.52
Test item concentrations: 7.84 - 8.78

Nominal and measured concentrations:

Control, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (nominal)
Control, 0.549, 0.762, 1.15, 1.67 and 1.68 mg/L (geometric mean measured conc)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: Sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, filled with 100 mL of test or control medium, headspace of 150 mL, flasks were capped with foam bungs.
- Initial cells density: starting alga cell concentration of 1 x 10E^04 cells/mL
- Control end cells density: 134 x 10E^04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Stock solutions for Alga culture medium contained various nutrients which were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The stock solutions/alga culture medium were sterilised by autoclaving or filtering (0.2 m filter pore size) as appropriate.
- Metals: FeCl3.6H2O: 0.064 mg/L
- Ca/mg ratio: MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: ph: at test start and test end; Temperature: continously (controlled incubator)

OTHER TEST CONDITIONS

- Adjustment of pH: No
- Photoperiod: Continously
- Light intensity and quality: Light intensity at 0 hours: 5890 – 7220 Lux; Light intensity at 72 hours: 5740 – 7280 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: Control; 0.1; 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: 0%, -26%, 1% and 95% (inhibition of growth rate); based on these results the test item concentrations for the main test were prepared

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits

Remarks:

0.805 – 1.34 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.762 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: after 72 h the Control, 0.625 and 1.25 mg/L: algae cells were bright green colour, sickle shaped; at 2.5 mg/L:Cells were bright green, mainly sickle shaped with a few flat cells; at 5.0 and 10 mg/L: cells were bright green, mainly sickle shaped with a few flat cells,
- Colour differences: No
- Flocculation: some flocculation of cells at 5 and 10 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.84 mg/L for growth rate
- Other: NOEC for growth rate was determined to be at 0.32 mg/L

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8.
Average specific growth rate: 0-24 hour, 0-48 hour and 0-72 hour. In addition, 24-48 hour and 48-72 hour growth rate was also assessed. The no observed effect concentrations (NOEC) were determined using the following tests;
Dunnett Multiple Comparison Test: 0-24, 24-48 and 48-72 hours average specific growth rate. Williams Multiple Comparison Test: 0-48 and 0-72-hour average specific growth rate and 72-hour yield.
The EC10, EC20 and EC50 values for the 72-hour final yield, the 0-24, 0-48, 0-72, 24-48 and 48-72 hour average specific growth rate (μ) time intervals were estimated using linear interpolation.
To distinguish between ECx values, estimated using final yield and growth rates, the symbols EyCx and ErCx were used, respectively.

For presentation purposes, some numerical data may have been rounded; therefore, manual recalculation may result in slightly different values to those presented in the results section.

Biological Results

Table 1: Inhibition of growth rate in %

Nominal concentration (mg/L)	Geometric mean measured concentration (mg/L)	Reduction in average specific growth  rates relative to the control (%)
		0-24 hours	0-48 hours	0-72 hours	24-48 hours	48-72 hours
Control	Control	-	-	-	-	-
0.625	0.549	0	-3	2	-5	10
1.25	0.762	-2	5	3	11	0
2.5	1.15	18	6	12	-4	22
5.0	1.67	44	32	27	22	19
10	1.68	49	33	33	18	35
-	Not applicable
Negative percentage inhibition values relative to the control represent an increase in growth relative to the control

Table 2: Endpints

Parameter		Geometric mean measured concentration (mg/L)
		72-Hour
Yield	EyC10	0.645 (NC – 0.950)
	EyC20	0.832 (NC – 0.997)
	EyC50	1.23 (0.941 – 1.46)
	NOEC	0.762
Growth Rate	ErC10	1.05 (0.805 – 1.34)
	ErC20	1.40 (1.19 – 1.55)
	ErC50	>1.68
	NOEC	0.762

 (x – x) = 95% confidence limits; NC = Not calculated

Table 3: Cell Growth of Raphidocelis subcapitata during the Definitive Test

Nominal concentration (mg/L)	Geometric mean measured concentration (mg/L)	Cell Concentration (cells/mL)			Mean Cell Concentration (cells x 104/ mL)
		24-hour	48-hour	72-hour	24-hour	48-hour	72-hour
Control	Control	18010*	79000*	366220*	4.33	21.9	134
		47280	233140	1225760
		45460	225940	1490820
		39000	119920	1167140
		38690	258540	1289400
		46250	255060	1504520
0.625	0.549	32780	171560	810560	4.40	23.4	125
		45730	252220	1441640
		53560	277440	1490700
1.25	0.762	50950	192120	1126200	4.45	18.6	115
		39430	220100	1146300
		43190	144480	1181920
2.5	1.15	25320	136100	721060	3.39	17.9	73.8
		34830	162800	592460
		41500	236960	899880
5.0	1.67	22200	74740	365480	2.29	7.91	35.2
		20730	68920	303120
		25800	93640	388620
10	1.68	18500	77040	303120	2.11	7.78	26.1
		21080	69500	210500
		23700	86900	270440

 * Replicate not included in results calculation as shown to be a statistical outlier (Grubbs Extreme Value test)

Analytical Results of the main test

Table 4: Chemical analysis during the Definitive Test

Nominal concentration (mg/L)	Measured concentration (mg/L)				Geometric mean measured concentration(mg/L)
	0 hours (new media)	24 hours (old media)	48 hours (old media)	72 hours (old media)
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
0.625	0.731 (117)	0.650 (104)	0.579 (93)	0.330 (53)	0.549 (88)
1.25	1.05 (84)	0.920 (74)	0.751 (60)	0.464 (37)	0.762 (61)
2.5	1.76 (70)	1.46 (58)	1.07 (43)	0.631 (25)	1.15 (46)
5.0	2.74 (55)	2.01 (40)	1.43 (29)	0.990 (20)	1.67 (33)
10	2.68 (27)	2.32 (23)	1.45 (15)	0.877 (9)	1.68 (17)

LOQ = Limit of quantification of the analytical method (0.001 mg/L)
Values in brackets represent % nominal value. However, it should be noted that the nominal concentrations of 5.0 and 10 mg/L were in excess of the solubility of the test substance

Table 1: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	133	Yes
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 must not exceed 35%	15.5
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2.35

Summary of results from the range-finding test

 

Specific growth rate and % inhibition relative to control

Nominal concentration (mg/L)	0 to 72 hours (x 10-2 cells/mL)	% Inhibition relative to control
Control	3.971	NA
0.10	5.022	-26
1.0	3.947	1
10	0.209	95

NA – Not applicable
Negative percentage inhibition values relative to the control are indicative of stimulated growth and are not considered to be an adverse effect

The biological results from the range-finding test differed from those obtained in the definitive test in that 95% reduction in growth rate was observed at the highest concentration in the range-finding test compared to 33% observed in the definitive test. The highest concentration was prepared from an initial 10 mg/L loading rate which was in excess of the solubility of the test substance in test media. The measured concentration at the highest concentration during the range-finding test was ca 30% greater than that observed during the definitive test (this is not unusual when the test substance is prepared from an excess of material) which could have resulted in increased growth inhibition. The definitive test is considered to be the more robust test given that increased replication was used and cell counts were performed daily giving more statistical power to the results analysis.

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErC50 (72h): >1.68 mg/L (Raphidocelis subcapitata, growth rate, based on the geometric mean measured concentration)

ErC10 (72h): 1.05 mg/L (Raphidocelis subcapitata, growth rate, based on the geometric mean measured concentration)

Additional information

One experimental study was available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to the OECD 201 (GLP) using freshwater algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (Goodband, 2020). The static test was carried out with the test item concentrations of control, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (based on the nominal concentrations) and control, 0.549, 0.762, 1.15, 1.67 and 1.68 (based on the geometric mean measured concentration). At the start of the test (0 hours) the measured concentrations were shown to be 0.731, 1.05, 1.76, 2.74 and 2.68 mg/L (117, 84, 70, 55 and 27% of nominal, respectively). After 72 hours, the measured concentrations were shown to be 0.330, 0.464, 0.631, 0.990 and 0.877 mg/L (53, 37, 25, 20 and 9% of nominal, respectively).

Given that the analytical results showed a decline in measured concentration over the 72-hour test period, the results have been calculated based on geometric mean measured concentrations. The geometric mean measured concentrations were calculated to be 0.549, 0.762, 1.15, 1.67 and 1.68 mg/L (88, 61, 46, 33 and 17% of nominal, respectively). Based on geometric mean concentrations, the 72-hour EyC50 and ErC50 values were estimated to be 1.23 and >1.68 mg/L, respectively. The corresponding EC10 value for yield and growth rate were considered to be 0.645 and 1.05 mg/L. All validity criteria were met, therefore, the test was considered valid.