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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:
31-07-2019 to 27-11-2019
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:
July 2011
Deviations:
yes
Remarks:
See 'Overall remarks, attachments'
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
N/A – study was conducted according to standard test guidelines
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Purity: 99.7096 area-% (Analysis)
Homogeneity: Homogeneous
Expiry Date: 07 Mar 2021
Storage conditions: Room temperature
Analytical monitoring:
yes
Details on sampling:
Samples (8 ml) were taken at the start of exposure (0 h) from the uninoculated replicates 7 and from inoculated replicates 8 of each test group. At the end of the exposure (72 h) samples were taken from the uninoculated replicates from each test group and from the combined inoculated replicates from each test group. For each test group, two retained samples were taken in plastic tubes at each time point. The retained samples were stored in a freezer and analysed if necessary or discarded after termination of the study. Retained samples were described by the suffix “b” and “c” in the study report. Unless frozen, samples were transported to the analytical laboratory on the day of sampling.
Vehicle:
no
Details on test solutions:
Test substance was first homogenised by shaking the container before use. Stock test solution (11.1 mg/L) was prepared by directly adding 33.3 mg of test substance to a 3 L plastic vessel of test medium and stirring for approximately 10 minutes on a magnetic stirrer at 900 rpm until visibly dissolved. The stock solution appeared colourless-clear. The lower test concentrations were prepared in plastic beakers by dilution of the stock solution (dilutions were stirred for approximately 5 minutes) and were selected on the basis of a preliminary range finding test. Before dilutions were performed, the stock solution was checked for complete dissolution of the test substance. Due to possible adsorption of the test item, test vessels were saturated with test solution for approximately 1 day before the start of exposure. Erlenmeyer flasks (nominal volume 250 mL) were completely filled without headspace and closed with glass plugs as the test substance is potentially volatile. Before exposure, the used solutions for saturation of test vessels were exchanged by freshly prepared test solutions. The test solution pH was within the acceptable ±0.5 range of the control.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algal test organism, Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81), were obtained from the SAG (Collection of algal cultures in Göttingen, Germany).

The stock algal culture was maintained continuously at the test facility. Before exposures, an inoculum culture of the test organism was prepared, with an aliquot of the stock algal culture added to sterile OECD media to provide an initial cell density of 0.3 x 10^4 cells/mL in the test replicates. The inoculum culture was incubated under test conditions for 4 days prior to test initiation. The increase in biomass was verified to ensure that growth was within the normal range and algal cells were examined microscopically for normal morphology prior to use for test inoculation. After this time, the inoculum culture was in the exponential growth phase and could be used to initiate the test (study day 0).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
No further remarks
Post exposure observation period:
N/A
Hardness:
Not specified
Test temperature:
23.3 - 23.4°C
pH:
7.4 to 10.8 (The increase in pH did not affect control algal growth since all validity criteria were fulfilled)
Dissolved oxygen:
N/A
Salinity:
N/A
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.1, 0.32, 1.0, 3.2, 10 mg/L (based on test substance mass without correction for purity)
Details on test conditions:
Test medium was prepared according to OECD 201 guideline), with the exception that in order to accommodate testing in a closed system, the NaHCO3 concentration in the final modified medium was increased to 300 mg/L and adjusted to pH 7 by the addition of 1M HCl. The test medium was sterilised after preparation.

Tests were conducted in Erlenmeyer flasks (nominal volume 250 mL) closed with glass plugs, with flasks completely filled with test solution to ensure no headspace (given that the test solution is potentially volatile). Tests included 6 replicates for the control and 3 replicates per test concentration. One additional uninoculated (without algae) (replicate 7) and inoculated (replicate 8) replicate were also included per test group for initial concentration control analysis. One additional uninoculated (without algae) replicate per test group was also included for background fluorescence correction (replicate 0) and to determine the influence of the test design on test substance stability.

Prior to the introduction of test organisms to test vessels, the cell density of the algal inoculum culture in the exponential growth phase was determined and then adjusted to 3 x 10^4 cells/mL with OECD media, so that a 1:10 dilution of inoculum resulted in an initial cell density of 0.3 x 10^4 cells/mL in the test replicates. The initial inoculum biomass was reduced in this test due to the closed test vessels. The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

Test vessels were maintained in a Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet throughout the testing period. Test vessels were placed under artificial light (type universal white (OSRAM L 25)) throughout the entire testing period, and test vessels rearranged daily to minimise potential effect of slight variations in illumination. Light intensity was 5476.4 ± 145.21 lux (within ± 15% variability) at a wavelength of 400 - 700 nm and was adjusted to the lower end of the acceptable range, to control pH changes due to excessive algal growth in the closed test vessels. Test solutions were stirred continuously (approximately 130 rpm) or twice daily by hand during the test period.

Algal growth was measured at 0, 24 and 72 h as in vivo chlorophyll-a fluorescence. Fluorescence of aliquots from each test vessel was measured using a Tecan Infinite 200Pro fluorometer in a 96-well flat bottom black plate. Samples of test solution were taken for pH measurement at the end of exposure (72 h) for replicates, but also at the start of exposure (0 h) for replicates 0 and 1. Temperature was continuously measured during the whole test period in the climate chamber in a separate deionised water filled flask. Light homogeneity was evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of the test (Testo 545 light meter, Testo GmbH & Co, Lenzkirch, Germany). The light intensity did not vary by more than ±15% over the incubation area.

At the end of exposure, the control replicates were mixed and serially diluted by a factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer) and these data used to derive a linear correlation between fluorescence and cell density.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorphenol
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: (95% confidence limits: 0.183 - 0.229)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: (95% confidence limits: 0.274 - 0.325)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: (95% confidence limits: 0.582 - 0.65)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% confidence limits: 0.463 - 0.649)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% confidence limits: 0.917 - 1.172)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% confidence limits: 3.242 - 3.791)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
All measured concentrations of the test substance in the test water deviated less than 20% from the nominal concentration. The test substance was therefore stable in solution under test conditions, and results were therefore evaluated based on the nominal concentrations. Additionally, samples were analysed from the uninoculated replicate 0 of each test concentration and the control at the end of the exposure (72 h). All uninoculated replicates were within ±20% of the nominal concentrations. All test solutions were visibly clear and colourless after preparation, with no visible undissolved test substance and no other remarkable observations through the end of exposure (72 h).

All validity criteria required by the corresponding guidelines were met; the cell multiplication factor in the untreated control was 648-fold after 72 hours (i.e. >16 after 72 hours), the mean coefficient of variation for section-by-section growth rates for each test day in the control cultures was 31.2% (i.e. ≤35%), and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 3.6% (i.e. ≤7%).
Results with reference substance (positive control):
The ErC50 (72 h) of the reference substance 3,5 Dichlorphenol was 2.074 mg/L, which falls within the typical ErC50 (72 h) range of 0.9 – 4.4 mg/L for the algal species tested.
Reported statistics and error estimates:
The mean fluorescence was determined after 0, 24, 48, 72 h and converted to cell density using a correlation curve derived from a microscopic count of the control. The commercial software "TOXRAT Professional 2.10” (ToxRat Solutions GmbH, Alsdorf, Germany) was used for the statistical evaluation of the data. The yield and specific growth rate over the exposure period was calculated for each replicate flask of each test group and inhibition for each test group was determined by comparison to the control. The percent inhibition of the mean yield and growth rate compared to the control was calculated for each test group replicate.

ECx values and confidence limits were calculated by probit analysis (Finney, 1971). The LOEC was determined by comparing the means of the calculated yield or growth rate of the various concentration levels with the control using Dunnett’s multiple t-test (one-sided). The NOEC was the next tested concentration below the LOEC. The yield and growth rate over the exposure period was calculated for each replicate flask of each test group (ISO/TS 20281) and inhibition for each test group was determined by comparison to the control. The percent inhibition of the mean yield and growth rate compared to the control was calculated for each test group.

Summary of analytically measured concentrations of the test substance in inoculated test solutions

Nominal concentration [mg/L]

Measured concentration in inoculated samples

0-h

72-h

mg/L

% of nominal

mg/L

% of nominal

0 (control)

<LoQ

-

<LoQ

-

0.1

0.108

108

0.093

93

0.32

0.33

103

0.301

94

1.0

1.029

103

1.009

101

3.2

3.198

100

3.224

101

10

10.334

103

10.396

104

 

Summary of analytically measured concentrations of the test substance in uninoculated test solutions

Nominal concentration [mg/L]

Measured concentration in uninoculated samples

0-h

72-h

mg/L

% of nominal

mg/L

% of nominal

0 (control)

<LoQ

-

<LoQ

-

0.1

0.1

100

0.112

112

0.32

0.317

99

0.338

106

1.0

1.009

101

1.039

104

3.2

3.104

97

3.236

101

10

10.315

103

10.124

101

 

 

Algal growth inhibition results based on nominal concentrations

Test Group (nominal) [mg/L]

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

at 72 hours

Mean yield

at 72 hours

% Inhibition

(as yield)a

Mean specific growth rate at 72 hours

% Inhibition

(as growth

rate)a

0 (control)

139

139

0

2.16

0

0.1

153

153

-10

2.21

-2.5

0.32

108

107.8

22

2.09

3

1.0

39.4

39.2

72

1.76

18

3.2

5.6

5.4

96

1.08

50

10

1

0.8

99

0.55

75

a: Negative values indicate a stimulatory effect relative to the control.

Numbers inboldare statistically significantly different from Control *p≤0.05

 

 

Algal morphology observations recorded after 72 hours

Test Group (nominal)

[mg/L]

Microscopic observations

0 (control)

No remarkable observations

0.1

No remarkable observations

0.32

No remarkable observations

1.0

No remarkable observations

3.2

Algal cells were hardly observed

10

Algal cells were hardly observed

 

  

Dissolution behaviour

Test Group (nominal)

[mg/L]

0 hour

24 hours

48 hours

72 hours

0 (control)

0

0

7

7

0.1

0

0

7

7

0.32

0

0

7

7

1.0

0

0

7

7

3.2

0

0

7

7

10

0

0

0

0

0: no remarkable observations

7: other: clear test solution with green coloration (sign for algal growth)

Validity criteria fulfilled:
yes
Remarks:
The test was compliant with all validity criteria required by the corresponding guidelines are is considered valid
Conclusions:
The 72-hour algal yield EyC50 was 0.6 mg/L (95% confidence limits: 0.582 – 0.65), the NOEyC was 0.1 mg/L and the LOEyC was 0.32 mg/L. The 72-hour algal growth rate ErC50 was 3.5 mg/L (95% confidence limits: 3.242 – 3.791), the NOErC was 0.32 mg/L and the LOErC was 1.0 mg/L.
Executive summary:

The toxicity of N,N-Dimethylcyclohexylamin to the algae Pseudokirchneriella subcapitata was determined. The study was run with nominal concentrations of 0 (control), 0.1, 0.32, 1, 3.2, 10 mg/L under static conditions in accordance with the OECD 201 guideline. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The 72-hour algal yield EyC50 was 0.6 mg/L (95% confidence limits: 0.582 – 0.65), the NOEyC was 0.1 mg/L and the LOEyC was 0.32 mg/L. The 72-hour algal growth rate ErC50 was 3.5 mg/L (95% confidence limits: 3.242 – 3.791), the NOErC was 0.32 mg/L and the LOErC was 1.0 mg/L. The test was compliant with all validity criteria required by the corresponding guidelines are is considered valid.

Description of key information

The toxicity of N,N-Dimethylcyclohexylamin to the algae Pseudokirchneriella subcapitata was determined according to the OECD 201 guideline (Wigh and Wagner, 2019). The study was run with nominal concentrations of 0 (control), 0.1, 0.32, 1, 3.2, 10 mg/L under static conditions. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The 72-hour algal growth rate ErC50 was 3.5 mg/L (95% confidence limits: 3.242 – 3.791), the NOErC was 0.32 mg/L and the LOErC was 1.0 mg/L. The 72-hour algal yield EyC50 was 0.6 mg/L (95% confidence limits: 0.582 – 0.65), the NOEyC was 0.1 mg/L and the LOEyC was 0.32 mg/L. The test was conducted under GLP and was compliant with all validity criteria required by the corresponding guideline are is considered valid and reliable. 

Key value for chemical safety assessment

EC50 for freshwater algae:
3.5 mg/L
EC10 or NOEC for freshwater algae:
0.32 mg/L

Additional information

The key study to address the toxicity to aquatic algae and cyanobacteria exposed Pseudokirchneriella subcapitata to the following nominal concentrations: 0 (control), 0.1, 0.32, 1, 3.2, 10 mg/L. The following results were obtained:

Based on algal growth rate:

72 -hour ErC50: 3.5 mg/L (95% confidence limits: 3.242 - 3.791)

72 -hour NOErC: 0.32 mg/L

72 -hour LOErC: 1.0 mg/L

Based on algal yield:

72 -hour EyC50: 0.6 mg/L (95% confidence limits: 0.582 - 0.65)

72 -hour NOEyC: 0.1 mg/L

72 -hour LOEyC: 0.32 mg/L