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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:
From Mar. 07, 2013 to Oct. 24, 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
. Study well documented, GLP. The deviations are about the measured concentrations which decreases during the test. However it is due to the properties of the substance (not stable) and to the conditions of the algae test (static test).
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
(the test substance rapidly degraded and the measured concentration at the end of study was
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD principles of GLP
Specific details on test material used for the study:
The test substance was Colipa A050 (CAS #54381-16-7; Batch Number 20110103), also known as G11-A11117 (Blauentwickler); 4-(Di(2-hydroxyethyl)amino)-Anilin-sulfat (1:1), hydrat; 2,2`-[4-aminophyenyl))imino]bis(ethanol) sulphate; Ethanol, 2,2`-[(4-aminophenyl))imino]bis-,sulphate (1:1) (salt); 2-[[(4-aminophenyl)-(2-hydroxyethyl)]amino]ethanol sulphate; INCI name: N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate.
The Certificate of Analysis reported the active ingredient in the form of free base for Colipa A050 as 62.5% by weight. Consequently the data in this record are expressed as active ingredient in conformity with the study report.
Since the active ingredient is not the registered substance a new Certificate of Analysis was performed of the tested substance, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulphate monohydrate, with a purity level of 99.24%. So the final results of the study were re-calculated taking into account the revised purity level and the EC50 is expressed as the tested substance.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg a.i./L
- Sampling period: The concentrations of test substance were analysed at initiation (0 hour) and hours 2, 4, 8, 12, 24, 48, and 72 (termination).
(i) At 0 hours: 10 mL samples were collected from parent solutions.
(ii) At 2, 4, 8, 12, 24, and 48 hours: 10 mL samples were collected from additional replicates prepared for analytical sampling.
(iii) At 72 hours: 10 mL samples were collected after combining replicate solutions (A through F for the control and A through C for test substance treatments)
- Sample processing before analysis: Samples were centrifuged for 10 minutes at 3200 RPM. 9.0 mL aliquot from each sample was removed and diluted to 10 mL with 0.2% erythorbic acid in HPLC grade water. The samples were further diluted, if necessary, with 0.2% erythorbic acid in HPLC grade water to provide final concentrations within the analytical standard concentration range (i.e., 0.0150 to 0.600 mg a.i./L)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Primary standard: 0.0160 g (0.0100 g corrected for purity) of test substance was transferred to 500 mL volumetric flask and made up to the volume with dilution medium for a concentration of 0.020 mg/mL.
- Test solutions: Aliquots of the primary standard were diluted with dilution medium to a volume of 1.2 L to prepare 0.20, 0.63, and 2.0 mg a.i./L test solutions. 2.0 mg a.i./L test solution was diluted 1.2 L with dilution medium to prepare 0.019 and 0.061 mg a.i./L test solutions
- Controls: Test medium was used as control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: Not reported
- Source: Parent stock (Selenastrum capricornutum) was obtained from the Department of Botany, Culture Collection of Algae, University of Texas at Austin (UTEX)
- Age of inoculum: 4 days old
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled (24 ± 2° C) environmental chamber under continuous light (approximately 8,600 lux). Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.3 to 23.9°C
pH:
At 0 hours: 7.6
At 72 hours: 7.9 to 9.0
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
Nominal Concentrations: 0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg a.i./L
Measured concentrations of test substance at initiation were 93 to 110% of the nominal concentrations, indicating that the solutions were properly dosed. Measured concentrations of Colipa A050 decreased at successive time points, ranging from 24-Hour Time Weighted Concentrations: 24-Hour Time Weighted Concentrations (Estimated) : 72-Hour Time Weighted Concentrations: The measured concentration at 72 hours represented recoveries of 7 to 11% of the nominal concentrations for the 0.20, 0.63, and 2.0 mg/L treatments. One half of MQL (i.e., 0.00835 mg a.i./L) was used for to calculate average calculations when the measured value was 72-Hour Time Weighted Concentrations (Estimated) : The mean concentrations of the 0.019 and 0.061 mg/L treatments at 24 and 72 hours were estimated based on the mean percent of nominal of the 24 and 72 hour TWA of the 0.20, 0.63 and 2.0 mg/L treatments at each time point, i.e., 22 and 9% respectively. Details on the measured concentrations and the % of recoveries were provided in the table 1 under ‘Any other information on materials and methods incl. tables’
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask with Teflon lined screw cap
- Material, size, fill volume: 125 mL flask filled with 140 mL test solution (with minimum headspace)
- Type of flow-through: Not reported
- Initial cells density: 5.0 × 10(3) cells/mL (Each replicate vessel inoculated with 1.0 mL of an algal concentrate containing approximately 7.0 × 10(5) cells/mL resulting in a final density of approximately 5.0 × 10(3) cells/mL for each flask)
- Control end cells density: 70.3 × 10(4) cells/mL
- No. of vessels per concentration: 3 (replicates A, B, and C)
- No. of vessels per control: 6 (replicates A, B, C, D, E, and F)
Four additional replicates (replicates G to J for the control and D to G for test substance) per treatment were prepared and inoculated for analytical sampling at intermediate time points.
An additional replicate (replicate H) of the 0.019 mg a.i./L test substance treatment was also prepared and used to evaluate the potential for incorporation of the test substance into the algal biomass.

INOCULATION: The replicates were inoculated with algae within 30 minutes after test solution preparation. Replicate of the 0.019 mg a.i./L test substance treatment was not inoculated with algae.

GROWTH MEDIUM
- Standard medium used: Freshwater algal nutrient medium (FWAM) supplemented with 500 mg of NaHCO3/L to minimize pH shift was used.
- Preparation: Prepared by the addition of appropriate reagent grade salts to autoclaved ABC reagent water and was supplemented with 500 mg of NaHCO3/L to minimize pH shift. After preparation, the medium was pH-adjusted to 7.5 ± 0.1 using 0.1 N HCl, and filtered through 0.45-μm Millipore filters
- Detailed composition if non-standard medium was used: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ABC reagent water, produced by passing reverse-osmosis water through a series of deionization tanks and then through a 0.2-μm filter.
Chemical characteristics of the ABC reagent water is provided in the ‘Appendix B’ of the study report.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Light intensity was measured daily. Temperature and pH in the test solutions were measured prior to distribution of the solutions to the test flasks and at 72 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity and quality: 7,997 to 8,140 lux; continuous white fluorescent light

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At 24, 48, and 72 hours (±1 hour), cell density was measured in all replicates of the controls, as well as replicates of each test substance treatment by direct microscopic counting with a hemacytometer.

RANGE FINDING STUDY
- Range finding study conducted: Yes
- Nominal test concentrations: 0 (control), 1.0, 5.0, 10, and 15 mg a.i./L.
- Results used to determine the conditions for the definitive study: Based on the results of range finding study, a nominal concentration range of 0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg a.i./L was selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.338 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 0.337-0.340 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI 0.060-0.072 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.272 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 0.270-0.273 mg/L
Details on results:
- The ErC50, EyC50 and ErC10 were initially calculated as 0.213, 0.0416 and 0.171 mg a.i./L, and they were recalculated as 0.338, 0.066 and 0.272 mg/l based on the tested substance and taking into account the updated certificate of analysis (see the explanations provided in "Specific details on test material used for the study").

RANGE FINDING STUDY:
- Appearance of test solutions: The 5.0 and 10 mg a.i./L test solutions were clear with a slight pink tint and the 15 mg a.i./L test solution was clear with a pink tint at initiation. At 72 hours, the 5.0, 10, and 15 mg a.i./L test solutions were clear with a copper tint and increasing color intensity with the increasing concentrations.
- Analysis of test concentrations: Measured concentrations of test substance at test initiation were 0.970 and 10.3 mg a.i./L, which represented recoveries of 97 to 103% of the nominal concentrations. They decreased at successive time points ranging from 91 and 95% of nominal at 2 hours to 3, and from 2% of nominal at 72 hours.
- Cell density: The control cell density at test termination was 66.0 × 10(4) cells/mL. The percent change in cell density at 72 hours, as compared to the control mean cell density, was -69, -98, -99, and -99% in the 1.0, 5.0, 10, and 15 mg a.i./L treatments, respectively.
MAIN TEST:
Appearance of test solutions:
- The control and all test substance solutions <2.0 mg a.i./L appeared clear and colorless, with no visible precipitates, surface films, or undissolved test substance at initiation and throughout the test.
- The 2.0 mg a.i./L test substance solution was clear and colorless at initiation, had a slight pink tint at 24 hours, and had a slight copper tint at 48 and 72 hours.
Exponential growth in the control: Yes, after 72 hours of exposure, mean cell density in control group was 70.3 x 10(4) cells/mL. This was an increase of 141 times over the initial inoculation density. There was significant growth inhibition relative to the control treatment during the definitive test.
Mean cell density: Values ranged from 5.52 × 10(4) to 72.0 × 10(4) cells/mL at nominal concentrations 2.0 and 0.019 mg a.i./L respectively.
Percent inhibition in algal growth at 72 hours: Values ranged from -2% to 92% at nominal concentrations 0.019 and 2.0 mg a.i./L respectively. Percent inhibition in growth rate from time zero to 72 hours, as compared to the control, ranged from 0% at the nominal concentrations of 0.019 and 0.061 mg a.i./L to 52% at the nominal concentration of 2.0 mg a.i./L.
Percent inhibition in yield at 72 hours: Values ranged from -2% to 93% at nominal concentrations 0.019 and 2.0 mg a.i./L respectively.
- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
All statistical analyses were performed with SAS software (Version 9.3 for windows). The ErC50 and EyC50 estimates were calculated using a logistic (sigmoid-shaped) model fit to the data with percent inhibition as the dependent variable and concentration as the independent variable. Further details are provided in the study report.

Table 1: Cell Density Values forPseudokirchneriella subcapitata during a 72-Hour Exposure to Colipa A050 (Study# 68338)

Nominal Concentration (mg a.i./L)

Mean Cell Density (cells/mL x 10(4))a

% Inhibitionb

24 Hours

48 Hours

72 Hours (%CV)

72 Hours

Control

2.35

12.1

70.3 (CV: 1)

-

0.019

2.52

11.9

72 (CV: 4)

-2

0.061

2.29

12.7

68.9 (CV: 3)

2

0.2

1.37*

11.2*

52.7* (CV: 4)

25

0.63

1.33*

3.82*

32.9* (CV: 9)

53

2

1.11*

0.741*

5.52* (CV: 13)

92

a - Mean cell density calculated using the individual replicate densities

b - Percent inhibition as compared to the control was determined at 72 hours using the following equation:

% inhibition = [(mean control cell density) - (mean treatment cell density)x 100]/mean control cell density

* Significant reduction in cell density as compared to the control (Dunnett’s test,p= 0.05).

 

Table 2: Growth Rate Values From Time Zero forPseudokirchneriella subcapitataduring a 72-Hour Exposure to Colipa A050 (Study# 68338)

Nominal Concentration (mg a.i./L)

Mean Growth Rate (cells/mL/hour)a

% Inhibitionb

0-24 Hours

0-48 Hours

0-72 Hours

0-72 Hours

Control

0.0644

0.0663

0.0687 (CV: 0)

---

0.019

0.0673

0.0661

0.069 (CV: 1)

0

0.061

0.0635

0.0674

0.0684 (CV: 1)

0

0.2

0.0418*

0.0647*

0.0647* (CV: 1)

6

0.63

0.0407*

0.0423*

0.0581* (CV: 2)

15

2

0.0332*

0.00814*

0.0333* (CV: 5)

52

a - Mean growth rate values calculated using the individual replicate growth rates

b - Percent inhibition as compared to the control was determined at 72 hours using the following equation:

% inhibition = [(mean control growth rate) - (mean treatment growth rate) x 100]/mean control growth rate

* Significant reduction in growth rate as compared to the control (Dunnett’s test,p= 0.05).

 

Table 3:Yield Values forPseudokirchneriella subcapitataDuring a 72-Hour Exposure to Colipa A050(Study# 68338)

 

Nominal Concentration (mg a.i./L)

Yield (cells/mL x 10(4))a

% Inhibitionb

0-24 Hours

0-48 Hours

0-72 Hours

0-72 Hours

Control

1.85

11.6

69.8 (CV: 1)

---

0.019

2.02

11.4

71.5 (CV: 4)

-2

0.061

1.79

12.2

68.4 (CV: 3)

2

0.2

0.867*

10.7*

52.2* (CV: 4)

25

0.63

0.830*

3.32*

32.4* (CV: 9)

54

2

0.610*

0.241*

5.02* (CV: 14)

93

a -Mean yield calculated using the individual replicate yield

b- Percent inhibition as compared to the control was determined at 72 hours using the following equation:

% inhibition = [(mean control yield) - (mean treatment yield) x 100]/mean control yield

* Significant reduction in yield as compared to the control (Dunnett’s test,p= 0.05).

Validity criteria fulfilled:
yes
Conclusions:
The 72 hour EC50 value of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate to Pseudokirchneriella subcapitata for inhibition of growth rate and yield were 0.338 and 0.066 mg/L, based on time-weighted average concentrations (TWA). The ErC10 is 0.272 mg/L.
Since the validity criteria for the growth rate of the controls are met (mean CV for 0-24, 24-48 and 48-72h specific growth rate < 35%, and the CV for the average growth rate during the whole period in replicates < 7%), then the ErC50 and the ErC10 calculated on the growth rate are considered as the most reliable results.
Executive summary:

The toxicity of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate toPseudokirchneriella subcapitata was determined in a 72 hours algae growth inhibition test, following the OECD 201 guideline under static conditions.

3 replicates were tested for each test concentration and 6 replicates for control.

Based on the results of range finding test, the definitive test was conducted at following nominal test concentrations:

0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg active ingredient/L (the active ingredient being the tested substance without sulphate and monohydrate). Measured concentrations at test initiation were 93 to 110% of the nominal concentrations which was indicative of proper dosing of test solutions. The concentrations decreased at successive time points ranging from <MQL (minimum quantifiable limit) to 86% of nominal at 2 hours, and from <MQL to 1% of nominal at 72 hours.

72-Hour Time Weighted Concentrations (TWC) were <MQL (control), <MQL, <MQL, 0.0171, 0.0464, and 0.214 mg a.i./L for 0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg a.i./L nominal concentrations respectively. One half of MQL (i.e., 0.00835 mg a.i./L) was used for to calculate average concentration when the measured value was <MQL for a time point. Because of the rapid degradation of the test material under the test conditions, mean concentrations for the 0.019 and 0.061 mg/L treatments resulted in values less than the MQL.

Endpoints evaluated were EC50of growth rate and yield over a period of 72 hours initialy expressed as a.i. (that means without taking into account the sulphate) and then re-calculate on the basis of the tested substance.

The test substance was found to inhibit the growth of freshwater green alga after 72 hours of exposure with the following effects :

EyC50: 0.066mg/L based onTime Weighted Concentrations (Inhibition of the yield),

ErC50: 0.338mg/L based onTime Weighted Concentrations (inhibition of growth rate)

ErC10: 0.272 mg/L based onTime Weighted Concentrations (inhibition of growth rate)

Since the validity criteria for the growth rate of the controls are met (mean CV for 0-24, 24-48 and 48-72h specific growth rate < 35%, and the CV for the average growth rate during the whole period in replicates < 7%), then the ErC50 and the ErC10 calculated on the growth rate are considered as the most reliable results.

This toxicity test is classified as acceptable and satisfies the guideline requirements for the OECD Guideline 201.

Description of key information

The 72 hour EC50 value of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate to Pseudokirchneriella subcapitata for inhibition of growth rate and yield were 0.338 and 0.066 mg/L, based on  time-weighted average concentrations (TWA). The ErC10 is 0.272 mg/L.

Since the validity criteria for the growth rate of the controls are met (mean CV for 0-24, 24-48 and 48-72h specific growth rate < 35%, and  the CV for the average growth rate during the whole period in replicates < 7%), then the ErC50 and the ErC10 calculated on the growth rate are considered as the most reliable results.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.338 mg/L
EC10 or NOEC for freshwater algae:
0.272 mg/L

Additional information

The toxicity ofN,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate toPseudokirchneriella subcapitatawas determined in a 72 hours algae growth inhibition test, following the OECD 201 guideline under static conditions.

3 replicates were tested for each test concentration and 6 replicates for control.

Based on the results of range finding test, the definitive test was conducted at following nominal test concentrations:

0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg active ingredient/L(the active ingredient being the tested substance without sulphate and monohydrate).Measured concentrations at test initiation were 93 to 110% of the nominal concentrations which was indicative of proper dosing of test solutions. The concentrations decreased at successive time points ranging from <MQL (minimum quantifiable limit) to 86% of nominal at 2 hours, and from <MQL to 1% of nominal at 72 hours.

72-Hour Time Weighted Concentrations (TWC) were <MQL (control), <MQL, <MQL, 0.0171, 0.0464, and 0.214 mg a.i./L for 0 (control), 0.019, 0.061, 0.20, 0.63, and 2.0 mg a.i./L nominal concentrations respectively.One half of MQL (i.e., 0.00835 mg a.i./L) was used for to calculate average concentration when the measured value was <MQL for a time point. Because of the rapid degradation of the test material under the test conditions, mean concentrations for the 0.019 and 0.061 mg/L treatments resulted in values less than the MQL.

Endpoints evaluated were EC50of growth rate and yield over a period of 72 hours initialy expressed as a.i. (that means without taking into account the sulphate) and then re-calculate on the basis of the tested substance.

The test substance was found to inhibit the growth of freshwater green alga after 72 hours of exposure with the following effects :

EyC50: 0.066mg/Lbased onTime Weighted Concentrations (Inhibition of the yield),

ErC50: 0.338mg/Lbased onTime Weighted Concentrations (inhibition of growth rate)

ErC10: 0.272 mg/Lbased onTime Weighted Concentrations (inhibition of growth rate)

Since the validity criteria for the growth rate of the controls are met (mean CV for 0-24, 24-48 and 48-72h specific growth rate < 35%, and the CV for the average growth rate during the whole period in replicates < 7%), then the ErC50 and the ErC10 calculated on the growth rate are considered as the most reliable results.