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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:
The study was conducted between 25 April 2016 and 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Not required
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not required, method to guideline
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 3-[[3-(Dimethylamino)propyl]amino]propanenitrile (TK2710)
Physical state/Appearance: Clear colorless liquid
Batch: AAE1131000
Purity: 98.7%
Expiry Date: 27 July 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Formulated hardener for structural composites
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture Medium
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.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1 °C.
pH:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH of the test and control preparations are shown in table 1 below.
Dissolved oxygen:
Not measured
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
6.25, 12.5, 25, 50 and 100 mg/L
Details on test conditions:
Range-Finding Tests
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial 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 (2.9 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.
The control group was maintained under identical conditions but not exposed to the test item.
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 – 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 results of the initial range-finding test showed significant inhibition of growth occurred at the test concentration of 100 mg/L where the 0-Hour pH was determined to be 10.1. In order to determine whether it was the highly alkaline nature of the test item which caused the inhibitory effects observed, a second range-finding test was conducted whereby the pH of the 100 mg/L stock solution was adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells.
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. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing a series of dilutions to give further stock solutions of 10 and 1.0 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.7 mL) to give the required test concentrations of 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.
The control group was maintained under identical conditions but not exposed to the test item.
Exposure conditions in the second range-finding test were the same as those in the initial test.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours from the initial range-finding test in order to determine the stability of the test item under test conditions. All 0-Hour samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L. It was considered appropriate to adjust the pH of the 100 mg/L stock solution prior to performing serial dilutions and inoculating with algal cells in order to demonstrate that it was not the highly alkaline nature of the test item which caused the inhibitory effects observed.

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. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing a series of dilutions to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (3.2 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 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.

Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.70 x 105 cells per mL. Inoculation of 500 mL of test medium with 3.2 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Data Evaluation
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass.
The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test item vessel was calculated .

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period.
For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated.

Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Statistical Analysis
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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
• The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):
yes
Remarks:
Positive control; potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 d
Dose descriptor:
EC20
Effect conc.:
36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Details on results:
Range-finding Tests
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L. Inhibitory effects were observed at 100 mg/L in both the initial test and the second range-finding test where the pH was adjusted to 7.5 prior to inoculation with algal cells. It was therefore considered that it was not the highly alkaline nature of the test item which was the cause of the inhibitory effects observed.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test. It was considered appropriate to adjust the pH of the 100 mg/L stock solution prior to performing serial dilutions and inoculating with algal cells in order to demonstrate that it was not the highly alkaline nature of the test item which caused the inhibitory effects observed.
Chemical analysis of the test preparations taken from the initial range-finding test at 0 and 72 hours showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 93% to 101% of nominal and so the results are based on nominal test concentrations only.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

The following results were determined from the data:
Inhibition of growth rate
ErC10 (0 - 72 h) : 27 mg/L
ErC20 (0 - 72 h) : 36 mg/L
ErC50 (0 - 72 h) : 62 mg/L; 95% confidence limits 55 - 70 mg/L

where ErCx is the test concentration that reduced growth rate by x%.
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). There were no statistically significant differences (P0.05), between the control, 6.25 and 12.5 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 12.5 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 25 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 14 mg/L
EyC20 (0 - 72 h) : 19 mg/L
EyC50 (0 - 72 h) : 30 mg/L; 95% confidence limits 27 - 33 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P0.05), between the control, 6.25 and 12.5 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 12.5 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 25 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 113 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.
Mean cell density of control at 0 hours : 5.00 x 103 cells per mL
Mean cell density of control at 72 hours : 5.63 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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 5% 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 6.25, 12.5, 25 and 50 mg/L, however cell debris and almost no intact cells were observed to be present in the test cultures at 100 mg/L.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.5 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, 6.25, 12.5 and 25 mg/L test cultures were observed to be green dispersions. The 50 mg/L test cultures were observed to be pale green dispersions whilst the 100 mg/L test cultures were observed to be clear colorless solutions.


Results with reference substance (positive control):
Positive Control
A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 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:
N/A
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on nominal test concentrations gave the following results:

Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 62 55 - 70 12.5 25
Yield 30 27 - 33 12.5 25
Executive summary:

Introduction

A study was perford 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.

Methods.

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be to range from 93% to 101% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

Response Variable

EC50(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

Growth Rate

62

55

-

70

12.5

25

Yield

30

27

-

33

12.5

25

Description of key information

The toxicity of Aradur 1019 to aquatic algae was determined according to OECD test guideline 201.

The results are reported as ErC50 (72h) 62 mg/l, and NOErC 12.5 mg/l/

Key value for chemical safety assessment

EC50 for freshwater algae:
62 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information