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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:
01 Feb 2016 to 04 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
July, 2011
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Chemical name: Zinc bis(diethyldithiocarbamate)
- Batch number: 50724904
- Purity: 99.12%
- Date of reception in the test facility: 19 October 2015
- Expiration date: 27 July 2017
- Water solubility: 1 mg/L (according to the SDS)
- Storage conditions: well-ventilated place, ambient temperature and obscurity
Analytical monitoring:
yes
Details on sampling:
Extra replicates (two replicates vessels at each test concentration except for the control) were added to the test in order to perform additional samplings for analysis at 24 hour intervals during the exposure period (one replicate was sacrificed at T24h, the second one at T48h). The test media prepared specifically for analysis of exposure concentrations were treated identically to those used for testing (i.e. each vessel were inoculated with ca 1.0 x 104 cells/mL of P. subcapitata and incubated under identical conditions).
One satellite non-inoculated vessel per test concentration (containing only dilution water and test item at the requested concentration, including a control) was also incubated under the same test conditions. The objective was to determine the influence of algae on the behavior of the test substance under the test conditions.
Vehicle:
no
Details on test solutions:
As the test item has a low water solubility and is known to be unstable in the test conditions, it falls into the category of a "difficult substance" as defined by the OECD Guidance Document No 23 (OECD, 2000); the method of preparation was thus based on the recommendations of this document: a stock solution was prepared at 10 mg/L loading rate and stirred during around 1 hour in order to achieve the saturation concentration, then diluted to get the required test solutions. Nondissolved particles were removed from the test medium per filtration through a 0.45 μm membrane filter.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Centre of Algae and Protozoa (Ambleside, UK).
- Age of inoculum (at test initiation):
- Method of cultivation: Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline. The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 - 23.1 °C
pH:
7.9 - 8.1
Dissolved oxygen:
8.4 - 8.9 mg/L dissolved O2
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 7.6, 12.2, 19.5, 31.3, 50.0 and 80.0 % v/v saturated solution
- Geometric mean measured concentrations: 0 (control), 18.4, 26.6, 46.9, 47.5, 103.1 and 226.8 µg/L. See 'Any other information on materials and methods incl. tables' for the daily measured concentrations in the saturated solutions.
Details on test conditions:
TEST SYSTEM
- Incubation chamber: The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions
- Test vessel: Glass bottles (120 mL capacity).
- Type: Closed; the test vessels were capped with cellulose bungs.
- Fill volume: 50 mL
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 1.6176E+06
- No. of vessels per concentration: 3
- No. of vessels per control: 6
Three days before the start of the exposure, pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density, i.e. 1E+04 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures.

GROWTH MEDIUM
- Standard medium used: The OECD medium recommended by the OECD Guideline No. 201 was the culturing medium for test organism and the diluent/solvent for the test item. Osmotically filtered water was used to prepare the dilution water. Chemical reagents used for the preparation of dilution water were of “analytical grade”.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: At the beginning of the test and in inoculated flasks at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: Constant illumination;
- Light intensity: 4900 - 5800 lux
- Light type: Fluorescent
Flasks were continuously shaken with a rotation at around 100 rpm

EFFECT PARAMETERS MEASURED: algal cell density
Algal cell concentrations were measured in each flask at 24, 48 and 72 h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. After 24, 48h and 72h of incubation, aliquot of 200 μL was sampled from each inoculated test flask and pipetted into a microplate. Time between sampling and measurement is approximately 15 - 30 min. The cytometer is calibrated every week using the Guava check kit. Before each measurement,
settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than alga were used to assess the toxic effects. The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell concentration was less than 500 cells/μL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore). Consequently, average specific growth rate inhibition and yield inhibition were calculated.

OTHER MEASUREMENTS: temperature, pH, dissolved oxygen
Dissolved oxygen concentrations and pH were measured in all test solutions, in non-inoculated flasks at the beginning of the test and in inoculated flasks at the end of the test. The temperature in the incubator was continuously recorded throughout the test.

INVALID ALGAL TEST (RANGE-FINDING)
A first algae test was conducted but invalidated as the test concentrations range was not arranged in a geometric series as recommended in the Guideline (one concentration was omitted by mistake). Nevertheless this test was finalized to estimate algae cell inhibitions although the test item concentrations were not determined.
- Test item filtrates: 6.0, 9.5, 15.3, 24.4, 39.1 and 100% v/v
- Results used to determine the conditions for the definitive study: 18.5% growth rate inhibition was observed in the 24.4% v/v filtrate. 100 and >100% growth rate inhibition were observed in the two highest filtrates, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
47.5 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
47.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
47.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
47.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
At exposure termination, in the 7.6, 12.2 and 19.5 % saturated solutions, no distinctive changes in growth rate from the control were observed. At a saturated solution of 31.3% (47.5 µg/L measured), growth rate inhibition was 48.1 %. At 50.0 and 80.0 % saturated solutions, > 100% inhibition of growth rate was observed. An overview of the effects of the test substance on inhibition is provided in 'Any other information on results incl. tables'.
- Test item solubility: 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, no precipitation was observed at the end of the test.
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 latest value of ErC50 obtained in October 2016 was 1.27 mg/L.
Reported statistics and error estimates:
The growth inhibition data were analysed using an Excel program. EC10 and EC50 values as well as the 95% confidence intervals were then determined using the software ToxRat® according to the recommendations of the guideline.

Table: Algal density

Test solution

Replicate

Algal density at 24h (Fd, ¢ *10E+04/mL)

Algal density at 24h (Fd, ¢ *10E+04/mL)

Algal density at 24h (Fd, ¢ *10E+04/mL)

Average specific growth rate d0-d3

Specific growth rate inhibition (%)

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Average

Sx

CV

Inhibition (per replicate)

Inhibition (mean)

Control

a

6.40

6.07

5.49

32.58

28.07

9.46

160.27

161.76

5.10

1.692

1.695

0.017

1.020

b

6.48

28.88

166.74

1.705

c

5.71

27.98

158.34

1.668

d

6.14

26.78

171.27

1.714

e

5.93

27.66

166.07

1.704

f

5.72

24.55

147.88

1.665

7.60

a

5.26

5.83

15.39

29.67

28.13

9.53

161.17

163.41

8.22

1.694

1.698

0.027

1.601

0.1

-0.2

b

5.35

25.03

151.25

1.673

1.3

c

6.86

29.68

177.82

1.727

-1.9

12.20

a

5.49

6.26

14.01

23.57

28.51

19.11

146.98

173.32

17.84

1.663

1.715

0.058

3.397

1.9

-1.2

b

7.21

34.36

207.37

1.778

-4.9

c

6.07

27.61

165.60

1.703

-0.5

19.50

a

7.54

7.23

4.00

32.23

28.27

12.75

184.28

165.42

12.02

1.739

1.701

0.041

2.387

-2.6

-0.4

b

6.97

27.41

144.67

1.658

2.2

c

7.16

25.17

167.30

1.707

-0.7

31.30

a

2.75

2.79

3.06

4.21

4.67

9.07

11.97

14.07

14.22

0.828

0.879

0.048

5.493

51.2

48.1

b

2.74

4.82

14.28

0.886

47.7

c

2.89

5.03

15.96

0.923

45.5

50.00

a

1.67

1.65

0.76

0.99

0.87

12.93

0.49

0.49

13.72

-0.240

-0.240

0.046

-19.122

114.1

114.1

b

1.64

0.85

0.56

-0.194

111.4

c

1.65

0.77

0.42

-0.285

116.8

80.00

a

1.43

1.42

0.48

0.03

0.04

25.35

0.01

0.02

37.03

-1.480

-1.380

0.118

-8.581

187.3

181.4

b

1.42

0.05

0.01

-1.410

183.2

c

1.42

0.04

0.02

-1.249

173.7

 

Table: Percentage of inhibition

Nominal concentration (% v/v saturated solution)

Geometric means measured concentration (µg/L)

Growth rate Iµi (%)

Yield Iyi (%)

7.6

18.4

-0.2

-1

12.2

26.6

-1.2

-7.2

19.5

46.9

-0.4

-2.3

31.3

47.5

48.1

91.9

50

103.1

>100

>100

80

226.8

>100

>100

Iμi: average percentage inhibition of growth rate

Iyi: average percentage inhibition of yield

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'

Description of key information

The 72-h ErC50 and 72 -h ErC10 were determined to be 47.5 µg/L and 47.4 µg/L in Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:
47.5 µg/L
EC10 or NOEC for freshwater algae:
47.4 µg/L

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (Arkema, 2017).

In this study, exponentially growing freshwater alga (Pseudomkirchneriella subcapitata) were exposed to nominal test substance saturation concentrations of 0 (control), 7.6, 12.2, 19.5, 31.3, 50.0 and 80.0% v/v for 72 hours under static conditions. The test was performed in 3 replicates per test concentration (6 replicates for the control). Test concentrations were analytically verified and determined to be 0 (control), 18.4, 26.6, 46.9, 47.5, 103.1 and 226.8 µg/L (geometric/other mean). At the start of the test and after 24, 48 and 72 hours exposure duration, algal cell concentrations were determined. At exposure termination, in the 7.6, 12.2 and 19.5 % saturated solutions, no distinctive changes in growth rate from the control were observed. At a saturated solution of 31.3% (47.5 µg/L measured), growth rate inhibition was 48.1 %. At 50.0 and 80.0 % saturated solutions, > 100% inhibition of growth rate was observed. Based on these findings, the 72-h EC50 values for growth rate and yield are determined at 47.5 µg/L and 47.4 µg/L, respectively. The 72 -h NOEC is 47.4 µg/L for both endpoints.

One additional growth inhibition study with freshwater algae is available for zinc diethyldithiocarbamate. The test is a non-GLP, OECD 201 guideline study with Chlorella pyrenoidosa without analytical monitoring (van Leeuwen et al., 1985a; van Leeuwen, 1986). The 96-h ErC50 value is 1.1 mg/L.