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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Principles of method if other than guideline:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000. was also followed in this study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
It was not possible to develop a stable analytical method for the specific analysis of the test item. It was consequently decided to analyse the Total Organic Carbon (TOC) concentrations as a surrogate measure.
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below from replicates without algae.
Frequency at t=0 h and t=72 h
Volume 40 mL
Storage Samples were stored in a refrigerator (2-8°C) until analysis.
Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
At the end of the exposure period, the replicates without algae were not pooled at each concentration before sampling. Instead, the individual replicates were used to provide the samples to be analysed and the reserve samples, respectively.

Test solutions

Vehicle:
no
Details on test solutions:
The batches of Mercaptamine tested were white powders with a purity of 98.8-99.4% and not completely soluble in test medium at the loading rates initially prepared.

For the range-finding test, preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman). After adjusting the pH from 9.5 to 8.6 with 1 M HCl (Merck, Darmstadt, Germany), the collected fraction was used as the highest test concentration. Test medium without test item was filtered as well and the pH adjusted from 8.0 to 8.6 using 1 M NaOH (Merck, Darmstadt, Germany). 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.

For the final test, preparation of test solutions started with loading rates individually prepared at 1.0, 3.2, 10, 32 and 100 mg t.i./L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Water Accommodated Fractions were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman). The pH of the filtrates resulting from the two highest loading rates was adjusted from 9.0-9.6 to 8.5 with 1 M HCl (Merck, Darmstadt, Germany). The pH of the filtrates resulting from the two lowest loading rates was adjusted from 8.2 to 8.5 using 1 M NaOH (Merck, Darmstadt, Germany). After the pH adjustments, the obtained solutions were used as test concentrations. Test medium without test item was filtered and the pH adjusted from 7.9 to 8.5 using 1 M NaOH (Merck, Darmstadt, Germany). Afterwards, the treated medium was used for the control group. 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.

Any residual volumes were discarded.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST SYSTEM
Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

FRESH WATER ALGAE CULTURE
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.
Light intensity: 60 to 120 µE/m2/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:
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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
21 - 22 °C
pH:
8.1 - 8.6
Nominal and measured concentrations:
WAF loading rates: 1.0, 3.2, 10, 32, 100 mg/L
Nominal TOC concentrations: 0.31, 1.0, 3.1, 10, 31 mg/L
Details on test conditions:
Test Procedure and Conditions
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.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 68 to 73 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.


Controls: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration,
6 replicates of the control,
2 extra replicates of each test group without algae for sampling purposes at the end of the test,
1 replicate of each test concentration without algae background for the treated solutions for the determination of cell densities.
Reference substance (positive control):
yes
Remarks:
not concurrent

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
Potassium dichromate:
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.

Any other information on results incl. tables

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Mercaptamine

WAF loading rate (mg t.i./L)

Mean

Std. Dev.

n

%Inhibition

Control

1.758

0.0097

6

 

1.0

1.698

0.0201

3

3.4#

3.2

1.593

0.0141

3

9.4#

10

1.214

0.0159

3

31*

32

0.671

0.0070

3

62*

100

0.360

0.0951

3

80*

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

Table 2: Growth Rate And Percentage Inhibition At Different Time Intervals

Mercaptamine

WAF loading rate (mg t.i./L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.796

 

1.898

 

1.580

 

1.0

3

1.746

2.8

1.880

0.92

1.466

7.2

3.2

3

1.734

3.4

1.729

8.9

1.316

17

10

3

1.493

17

1.186

38

0.963

39

32

3

1.059

41

0.691

64

0.263

83

100

3

0.000

100

0.070

96

1.011

36

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EL50 for growth rate inhibition (72h-ERL50) was 22 mg t.i./L with a 95% confidence interval ranging from 21 to 24 mg t.i./L. The 72h-NOEL for growth rate inhibition was 3.2 mg t.i./L based on biological relevance