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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:
14 October 2018 to 12 December 2018
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:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
- Frequency: at t = 0 h, t = 24 h and t = 72 h.
- Volume: 6.0 mL from the approximate centre of the test vessels.
- Storage: Samples were stored in a freezer (≤-15 °C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling, instead samples were taken from one vessel.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10 % of the saturated solution (SS) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 6.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15 °C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of test material tested was a clear colourless to light yellow liquid and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test material. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.
Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring to ensure maximum dissolution of the test material in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Any residual volumes were discarded.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24 °C. Light intensity was 60 to 120 µE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3 500 mg/L, K2HPO4 39.5 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3 20 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L and (NH4)6Mo7O24.4H2O 0.018 mg/L.

ACCLIMATION
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: Adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L, NaHCO3 300 mg/L, HEPES buffer 6 mmol/L, Hardness (Ca + Mg) 0.24 mmol/L (24 mg CaCO3/L), pH 7.1 ± 0.3.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
(Ca + Mg) = 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22 - 24 °C
pH:
7.4 - 8.1
Nominal and measured concentrations:
- Nominal: Solutions containing 0,1.0, 3.2, 10, 32 and 100 % of the SS, prepared at a loading rate of 100 mg/L
- Measured (TWA): 0, 0.45, 1.4, 4.2, 3.7, 11 and 40 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL, airtight closed with minimum headspace to prevent any loss of the test material due to volatilisation.
- Initial cell density: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3; 1 extra replicate of each test group for sampling purposes after 24 hours of exposure and 1 or 2 replicates of each test concentration without algae were also included in the study.
- No. of vessels per control (replicates): 6
- Incubation: Vessels were distributed at random in the incubator and repositioned daily. During incubation the algal cells were kept in suspension by continuous shaking.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L, NaHCO3 300 mg/L, HEPES buffer 6 mmol/L, Hardness (Ca + Mg) 0.24 mmol/L (24 mg CaCO3/L), pH 7.1 ± 0.3.
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, for all test concentrations and the control. Temperature of medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 87 to 89 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the 10 % SS test concentration and the control to observe for any abnormal appearance of the algae.

RANGE-FINDING STUDY
- Range finding study: A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
Three replicates of exponentially growing algal cultures were exposed to solutions containing 1.0, 10 and 100 % of the SS, prepared at a loading rate of 100 mg/L and to a control. Cell densities were recorded only at the end of the exposure period. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval. pH was only measured in the control and the highest test concentration. At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

INTERPRETATION
- Calibration Curve: Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Comparison of Average Growth Rates: The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (ln Xj - ln Xi) / (tj - ti) (day^-1)
Where:
µi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = [(µC - µT) / µC] x 100
Where:
%Ir = percent inhibition in average specific growth rate
µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

- Yield: The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = [(YC - YT) / YC] x 100
Where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

- Determination of the Average Exposure Concentrations
The average exposure concentrations were calculated as:
[24 x √(Ct = 0 x Ct = 24) + 48 x √(Ct = 24 x Ct = 72)] / 72 , being the Time Weighted Average (TWA) of the concentrations of the test material measured in the samples taken at the start (Ct = 0), after 24 hours (Ct = 24) and the end of the test (Ct = 72).

ACCEPTABILITY OF THE TEST
1. In the control, cell density increased by an average factor of at least 16 within the exposure period.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 %.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limit: 6.1 to 6.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidence limit: 3.2 to 7.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
RANGE-FINDING TEST
- Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield: Dose-related inhibition of algal growth rate and yield was found at the end of the test. An inhibition of 83 and 99 % was observed at the highest test concentration, respectively. Based on these results, samples taken from solutions containing 1.0 and 100 % of the SS were analysed. The measured concentrations at the start of the test were 0.77 and 84 mg/L, respectively. Measured concentrations were at the level of 91 and 50 % of initial at the end of the test. All test conditions were maintained within the limits prescribed by the study plan.

FINAL TEST
- Measured Test Material Concentrations: Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.67, 2.4, 6.3, 18 and 59 mg/L, respectively. During the exposure period, the concentrations decreased to 33 - 57 % of initial at the end of the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test material in test medium throughout the test.
- Inhibition of Growth Rate and Inhibition of Yield: Inhibition of growth rate and yield increased with increasing concentration of the test material from 1.4 mg/L upwards resulting in 100 % inhibition at the two highest test concentrations. Statistically significant inhibition of growth rate and yield was found at test concentrations of 4.2 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 10 % of the SS when compared to the control.

ACCEPTABILITY OF THE TEST
- In the control, cell density increased by an average factor of at least 16 within the exposure period: 197.
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 %: 18 %.
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %: 1.8 %.
Results with reference substance (positive control):
- Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL.
- The study met the acceptability criteria prescribed by the study plan and was considered valid.
- Potassium dichromate inhibited growth rate of this fresh water algae species at all concentrations tested.
- The EC50 for growth rate inhibition (72h-ErC50) was 1.05 mg/L with a 95 % confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72 h-ErC50 for the algal culture tested corresponds with this range. The EC50 for yield inhibition (72 h-EyC50) was 0.295 mg/L with a 95 % confidence interval ranging from 0.292 to 0.298 mg/L.
Reported statistics and error estimates:
- For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α = 0.05, one-sided, smaller) after trend analysis by contrasts (monotonicity of concentration/response).
- Calculation of ECx-values was based on probit analysis for growth rate and weibull analysis for yield using linear max. likelihood regression with the percentages of growth rate inhibition or the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test material.
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Growth Rate and Percentage Inhibition for the Total Test Period

Test Material TWA conc. (mg/L) 

Mean

Std. Dev.

n

% Inhibition

Control

1.760

0.0322

6

-

0.45

1.787

0.0551

3

-1.5

1.4

1.754

0.0412

3

0.38

4.2

1.587

0.0574

3

9.9*

11

0.000

0.0000

3

100*

40

0.000

0.0000

3

100*

* - effect was statistically significant.

 

Table 2: Yield and Percentage Inhibition for the Total Test Period

Test Material TWA conc. (mg/L) 

Mean

Std. Dev.

n

% Inhibition

Control

196.3

18.83

6

-

0.45

214.1

34.02

3

-9.1

1.4

192.6

24.03

3

1.9

4.2

116.9

20.01

3

40*

11

0.0

0.00

3

100*

40

0.0

0.00

3

100*

* - effect was statistically significant.

 

Table 3: Effect Parameters

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

1.4

3.7

4.4

6.2

lower 95%-cl

 

3.6

4.3

6.1

upper 95%-cl

 

3.8

4.5

6.3

Yield

Value

1.4

2.0

2.8

4.8

lower 95%-cl

 

0.64

1.4

3.2

upper 95%-cl

 

6.3

5.6

7.0

cl – confidence limit.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, the test material inhibited growth rate and yield significantly at TWA concentrations of 4.2 mg/L and higher.
The 72 h-EC50 for growth rate inhibition (ErC50) was 6.2 mg/L with a 95 % confidence interval ranging from 6.1 to 6.3 mg/L. The 72 h-EC50 for yield inhibition (EyC50) was 4.8 mg/L with a 95 % confidence interval ranging from 3.2 to 7.0 mg/L. The 72 h-NOEC for both growth rate and yield inhibition was 1.4 mg/L based on statistical significance.
Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201 under GLP conditions.

The objective of the study was to evaluate the test material for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

A Saturated Solution of the test material (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.

A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.67, 2.4, 6.3, 18 and 59 mg/L, respectively. During the exposure period, the concentrations decreased to 33 - 57 % of initial at the end of the test. The TWA concentrations were 0, 0.45, 1.4, 4.2, 11 and 40 mg/L.

Inhibition of growth rate and yield increased with increasing concentration of the test material from 1.4 mg/L upwards resulting in 100 % inhibition at the two highest test concentrations. Statistically significant inhibition of growth rate and yield was found at test concentrations of 4.2 mg/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of the present study, the test material inhibited growth rate and yield significantly at TWA concentrations of 4.2 mg/L and higher.

The 72 h-EC50 for growth rate inhibition (ErC50) was 6.2 mg/L with a 95 % confidence interval ranging from 6.1 to 6.3 mg/L. The 72 h-EC50 for yield inhibition (EyC50) was 4.8 mg/L with a 95 % confidence interval ranging from 3.2 to 7.0 mg/L. The 72 h-NOEC for both growth rate and yield inhibition was 1.4 mg/L based on statistical significance.

Description of key information

Under the conditions of the present study, the test material inhibited growth rate and yield significantly at TWA concentrations of 4.2 mg/L and higher.

The 72 h-EC50 for growth rate inhibition (ErC50) was 6.2 mg/L with a 95 % confidence interval ranging from 6.1 to 6.3 mg/L. The 72 h-EC50 for yield inhibition (EyC50) was 4.8 mg/L with a 95 % confidence interval ranging from 3.2 to 7.0 mg/L. The 72 h-NOEC for both growth rate and yield inhibition was 1.4 mg/L based on statistical significance.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.2 mg/L
EC10 or NOEC for freshwater algae:
1.4 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

A Saturated Solution of the test material (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.

A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.67, 2.4, 6.3, 18 and 59 mg/L, respectively. During the exposure period, the concentrations decreased to 33 - 57 % of initial at the end of the test. The TWA concentrations were 0, 0.45, 1.4, 4.2, 11 and 40 mg/L.

Inhibition of growth rate and yield increased with increasing concentration of the test material from 1.4 mg/L upwards resulting in 100 % inhibition at the two highest test concentrations. Statistically significant inhibition of growth rate and yield was found at test concentrations of 4.2 mg/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of the present study, the test material inhibited growth rate and yield significantly at TWA concentrations of 4.2 mg/L and higher.

The 72 h-EC50 for growth rate inhibition (ErC50) was 6.2 mg/L with a 95 % confidence interval ranging from 6.1 to 6.3 mg/L. The 72 h-EC50 for yield inhibition (EyC50) was 4.8 mg/L with a 95 % confidence interval ranging from 3.2 to 7.0 mg/L. The 72 h-NOEC for both growth rate and yield inhibition was 1.4 mg/L based on statistical significance.