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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:
This study was conducted between 25 August 2017 and 15 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EC No. 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: Jeffcat LE 30
- CAS Number: 65286-55-7
- Chemical Name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Batch: DR74271215
- Purity: 98.2%
- Physical state/Appearance: Clear colorless liquid
- Expiry Date: 30 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature in the dark


Analytical monitoring:
yes
Details on sampling:
- Sampling method: A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. The test samples (50 mL) were thawed with the aid of a waterbath. An aliquot of ammonia (50 µL) was added to the test sample then transferred to a 100 mL volumetric flask, and made to volume with methanol. Where necessary, the samples were diluted into the calibration range with diluent. To demonstrate the stability of the test item in test medium, volumes of test medium were spiked with the test item and the half-life was assessed. The test item (nominal 100 mg) was initially dissolved in methanol to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked samples at a concentration of 10 mg/L. Six replicates at this concentration level were prepared and subjected to the same treatment as the test samples at 0, 1, 2, 4, 6 and 24 hours after spiking.

- Sample storage conditions before analysis: All samples were stored frozen prior to analysis
Vehicle:
no
Details on test solutions:
Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L. An initial experiment conducted failed to achieve near nominal concentrations in the 0-Hour test preparations and as such was repeated.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (2.3 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1”C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1”C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

ACCLIMATION
- Acclimation period: non relevant
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
Temperature was maintained at 24 ± 1 °C.  The temperature within the incubator was recorded daily.
pH:
The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 8.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. The pH was measured using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 88% to 100% of nominal. Analysis of the 3.2, 10, 32 and 100 mg/L test preparations at 72 hours showed measured test concentrations to be near nominal, a slight decline in measured test concentration was observed in the 1.0 mg/L test preparation to 76% of nominal. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Nominal Test Concentration (mg/L) Geometric Mean Measured Test Concentration (mg/L) Expressed as a Percentage of the 0-Hour Measured Test Concentration (%)
1.0 0.82 82
3.2 2.9 91
10 9.2 92
32 32 100
100 93 93
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation.  
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.10 x 106 cells per mL.  Inoculation of 500 mL of test medium with 2.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- Control end cells density: 1.14 x 10^6 cells per mL
- No. of organisms per vessel: Six flasks each containing 100 mL of test preparation were used for the control and 3 flasks each containing 100 mL were used for each treatment group.  
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 72 hrs
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter.  The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Inhibition of Growth Rate
ErC10 (0 - 72 h): 9.7 mg/L
ErC20 (0 - 72 h): 15 mg/L
ErC50 (0 - 72 h): 29 mg/L; 95% confidence limits 23 - 37 mg/L

Inhibition of Yield
EyC10 (0 - 72 h): 2.2 mg/L
EyC20 (0 - 72 h): 3.3 mg/L
EyC50 (0 - 72 h): 6.7 mg/L; 95% confidence limits 5.7 to 8.0 mg/L

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 227 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.14 x 10^6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 15% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.82 and 2.9 mg/L. Clumped cells were observed to be present in the 9.2 mg/L test cultures whilst no intact cells were observed to be present in the 32 and 93 mg/L test cultures.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to range from pH 7.4 at 0 hours to pH 8.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.82 and 2.9 mg/L test cultures were observed to be green dispersions. Replicate R1 of the 9.2 mg/L test cultures was observed to be a slightly pale green dispersion whilst replicates R2 and R3 were observed to be pale green dispersions. The 32 and 93 mg/L test cultures were observed to be clear colorless solutions.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

NOErC: 0.25 mg/L
NOEyC: 0.25 mg/L
LOErC: 0.50 mg/L
LOEyC: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.075

-

1.09E+06

-

R2

0.075

1.12E+06

R3

0.075

1.14E+06

R4

0.076

1.21E+06

R5

0.076

1.15E+06

R6

0.075

1.09E+06

Mean

0.075

1.13E+06

SD

0.001

4.60E+04

0.82

R1

0.079

[5]

1.44E+06

 

R2

0.076

[1]

1.19E+06

 

R3

0.075

0

1.13E+06

 

Mean

0.077

[2]

1.25E+06

[11]

SD

0.002

 

1.68E+05

 

2.9

R1

0.070

7

7.79E+05

 

R2

0.072

4

8.84E+05

 

R3

0.074

1

1.01E+06

 

Mean

0.072

4

8.91E+05

21

SD

0.002

 

1.15E+05

 

9.2

R1

0.064

15

5.03E+05

 

R2

0.061

19

4.11E+05

 

R3

0.060

20

3.81E+05

 

Mean

0.062

18

4.32E+05

62

SD

0.002

 

6.39E+04

 

32

R1

0.023

69

2.11E+04

 

R2

0.013

83

7.53E+03

 

R3

0.012

84

6.65E+03

 

Mean

0.016

79

1.18E+04

99

SD

0.006

 

8.11E+03

 

93

R1

0.007

91

3.33E+03

 

R2

0.004

95

1.75E+03

 

R3

-0.002

103

-8.05E+02

 

Mean

0.003

96

1.42E+03

100

SD

0.005

 

2.09E+03

 

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R   = Replicate

SD   =Standard Deviation

-    = Not applicable

[ ] = Increase in growth as compared to the control

Validity criteria fulfilled:
yes
Remarks:
The data satisfied the validation criterion given in the OECD Guideline
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Response Variable EC50 (mg/L) 95% C.L. (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 18 15 - 21 2.9 9.2
Yield 6.7 5.7 - 8.0 0.82 2.9

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 88% to 100% of nominal. Analysis of the 3.2, 10, 32 and 100 mg/L test preparations at 72 hours showed measured test concentrations to be near nominal, a slight decline in measured test concentration was observed in the 1.0 mg/L test preparation to 76% of nominal.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. 

Based on the geometric mean concentrations, the 72-h EC50 based on growth rate was determined to be 18 mg/L with a 95% confidence interval ranging from 15 to 21 mg/L.

 

Key value for chemical safety assessment

EC50 for freshwater algae:
18 mg/L
EC10 or NOEC for freshwater algae:
2.9 mg/L

Additional information