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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:
07 Jan 2018 - 29 Jan 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:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test item information:
Identification: N-ethyl-o (or p) toluenesulphonamide (NETSA)
Appearance: Light yellow viscous liquid
Batch: 1703691015
Test item storage: At room temperature. Keep containers tightly closed in a dry, cool and well-ventilated place
Stable under storage conditions until: 16 March 2019 (expiry date)

Additional information:
Test Facility test item number: 201042/B
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: N-ethyl-o (or p) toluenesulphonamide
CAS number: 8047-99-2
Molecular formula: C9H13NO2S
Molecular weight: 199.3
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: 0.47 g/l
Stability in water: Stable
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 3.5 mL
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 pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate test item concentration (32% 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.
Vehicle:
no
Details on test solutions:
The batch of the substance tested was a light yellow viscous liquid with a purity of 100% and visually not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying an overnight period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle (combined limit/range-finding test: 181 minutes, final test: 229 minutes). 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 colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Analysis:
Stock and Spiking Solutions
Stock solutions of the test item were prepared in acetonitrile at concentrations of 1000 and 2000 mg/L.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was acetonitrile.
Calibration Solutions
Six working solutions in the concentration range of 0.05 - 20 mg/L were prepared in
30/70 (v/v) acetonitrile/ M2-medium from two stock solutions.
Quality Control (QC) Samples
3.5 mL blank medium was spiked with the test item at a target concentration of 0.1 or 100 mg/L. The QC samples were treated similarly as the test samples (see paragraph 2.2.2 ‘Test Samples’). Blank QC samples consisting of blank medium were treated similarly to the QC and test samples.

On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 7:3 (v:v) ratio with acetonitrile and analyzed.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata
Strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture : 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator varied between 22 and 23°C.
pH:
t=0h : 7.9 - 8.1
t =72h: 7.9 - 8.1
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).
Nominal and measured concentrations:
Nominal: Solutions containing 10, 18, 32, 56 and 100% of aqueous Saturated Solution (SS) prepared at a loading rate of 100 mg/L

The concentrations measured at the start of the test were 8.6, 16, 28, 51 and 88 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The initial concentrations remained stable throughout the test.
Details on test conditions:
TEST SYSTEM
- NETSA: Solutions containing 10, 18, 32, 56 and 100% of a SS prepared at a loading rate of 100 mg/L.
- Controls: Test medium without test item or other additives.
- Replicates:
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test concentration for sampling purposes after 24 hours of exposure
- 1 or 2 replicates of each test concentration without algae
- Test duration: 72 hours
- Test type: Static
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 10^4 cells/mL.

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 79 to 83 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENT AND RECORDINGS
- pH: At the beginning and at the end of the test.
- Temperature of medium: Continuously in a temperature control vessel.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the 18% SS test group and the control to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

COMBINED LIMIT/ RANGE- FINDING TEST:
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:
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a saturated solution prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• Three replicates per concentration were exposed to solutions containing 1.0 and 10% of the SS prepared at 100 mg/L.
• pH was only measured in the control and the highest test concentration.
• At the end of the test, algae were not observed to verify a normal and healthy appearance.

- Results used to determine the conditions for the definitive study: Yes. No inhibition of growth rates and yields was observed in the two lowest test concentrations during the 72-hour test period, whereas growth rate and yield were inhibited with 64 and 97%, respectively, in the undiluted SS. Based on these results samples taken from solutions containing 10 and 100% of the SS prepared at a loading rate of 100 mg/L were analysed. The initial concentrations were in agreement with the nominal concentrations throughout the test, i.e. were at 99-102% of the nominal concentrations. The expected EC50 for both growth rate inhibition and yield inhibition was thus between nominally 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 75-82 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 12-14
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance,
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological significance
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 14 - 15
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: Based on both statistical and biological significance
Details on results:
Measured Test Item Concentrations:
Samples taken from all test concentrations were analysed. The concentrations measured at the start of the test were 8.6, 16, 28, 51 and 88 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The initial concentrations remained stable throughout the test, i.e. were 101-104% of the initial concentration at the end of the test. The concentrations measured in samples with algae were comparable with the concentrations measured in the samples without algae. It was eventually concluded that the undissolved material which was removed during the preparation of test solutions was not test item related. Based on these results, effect parameters were expressed based on analytically confirmed nominal concentrations.

Inhibition of Growth Rate and Inhibition of Yield:
Inhibition of growth rate increased with increasing concentration of Ketjenflex 8 and was statistically significant at all test concentrations. Growth rate inhibition was considered to be biologically not relevant at the lowest test concentration of 10 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 10 mg/L. Inhibition of yield increased with increasing concentration of Ketjenflex 8 resulting in 96% inhibition at 100 mg/L and was both statistically significant and biologically relevant at all test concentrations. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 18 mg/L when compared to the control.

Acceptability of the Test:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 273 ).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 27%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.61%).
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOEC and the EC50 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 revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design inOECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of thetest item. No EC10 value could be estimated for yield inhibition (EYC10). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Growth Rate And Percentage Inhibition For The Total Test Period

NETSA

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.870

0.0114

6

10

1.734

0.0233

3

7.3*#

18

1.563

0.0044

3

16*

32

1.390

0.0295

3

26*

56

1.134

0.0392

3

39*

100

0.789

0.0440

3

58*

* Effect was statistically significant;#Effect was biologically not relevant (<10%).


Yield And Percentage Inhibition For The Total Test Period


Ketjenflex 8

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

272.4

9.37

6

 

10

181.1

12.92

3

34*

18

107.8

1.44

3

60*

32

63.9

5.87

3

77*

56

29.1

3.43

3

89*

100

9.7

1.37

3

96*

* Effect was statistically significant.

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, under the conditions of this study with Raphidocelis subcapitata, NETSA reduced growth rate and inhibited the yield of this fresh water algae species significantly at all concentrations tested. The 72h -NOErC, was < 10 mg/L when based on statistical analysis and 10 mg/L when based on biological relevance. ERC10 and ERC50 were 13 and 78 mg/L respectively. While the 72h-NOEyC and EYC50 were < 10 and 15 mg/L respectively.

Executive summary:

To assess the toxicity of NETSA to algae, a full OECDTG 201 GLP test was performed with Raphidocelis subcapitata based on the results of a preceding combined/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing the test substance under static conditions. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The concentrations measured at the start of the test were 8.6, 16, 28, 51 and 88 mg/L in solutions containing 10, 18, 32, 56 and 100% of the Saturated Solution prepared at a loading rate of 100 mg/L, respectively.

The 72h -NOErC was < 10 mg/L when based on statistical analysis and 10 mg/L when based on biological relevance,

ERC10 and ERC50 were 13 and 78 mg/L respectively. The 72h-NOEyC and EYC50 were < 10 and 15 mg/L respectively.

Description of key information

To assess the toxicity of NETSA to algae, a full OECDTG 201 GLP test was performed with Raphidocelis subcapitatabased on the results of a preceding combined/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing the test substance under static conditions. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The concentrations measured at the start of the test were 8.6, 16, 28, 51 and 88 mg/L in solutions containing 10, 18, 32, 56 and 100% of the Saturated Solution prepared at a loading rate of 100 mg/L, respectively.

The 72h -NOErC was < 10 mg/L when based on statistical analysis and 10 mg/L when based on biological relevance,

ERC10 and ERC50 were 13 and 78 mg/L respectively. The 72h-NOEyC and EYC50 were < 10 and 15 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
78 mg/L
EC10 or NOEC for freshwater algae:
13 mg/L

Additional information