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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
Study period:
21 October 2016 - 22 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.
Version / remarks:
14 December 2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
solid
Details on test material:
Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container
Specific details on test material used for the study:
- Irritant or corrosive: yes
- Solubility in water: 5-10%
- Stability in water: stable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 2.0 mL
Storage: Samples were stored in silanised vials in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method (for definitive test): No correction was made for the purity/composition of the test item. Test item was ground prior to preparation. Preparation of test solutions started with the highest concentration of 1.0 mg/L applying 17-25 minutes of magnetic stirring to accelerate the dissolution of the test item in test medium. This resulted in a clear and colorless solution. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colorless.
Glassware was incubated with test concentrations 24 hours prior to the start of the test to improve the stability of the exposure concentrations, as the test item tended to absorb to glass. Test solutions used for the incubation of the glassware were discarded prior to the start of the test and algal cells were exposed to freshly prepared solutions.
- Controls: test medium without test item or other additives (blank-control).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.

CULTIVATION:
- Method: 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
- Medium different from test medium: yes, M1

- Pre-culture: 3-4 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 different from test medium: for the definitive test, the algae were not pre-cultured but used directly from the stock.

ACCLIMATION
- Acclimation period: no

Study design

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

Test conditions

Hardness:
24 mg CaCO3 mg/L
Test temperature:
22 - 23°C
pH:
At the start of the test: 7.9-8.0
At the end of the test: 8.0-8.1
Nominal and measured concentrations:
Nominal test concentrations: 0.32, 1.6, 8.0, 40, 200 and 1000 µg/L
Measured test concentrations: see table 1
As actual exposure concentrations could not be reliably calculated based on the analytical results, it was decided to express the effect parameters in terms of nominal concentrations. See 'Overall remarks' for justification of using nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean): 110.8 x 10^4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 or 2 replicates of each concentration without algae; 1 or 2 replicates for each test concentration for sampling purposes at t=24 h.

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 81 to 84 µE/m^2/s
- 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.

EFFECT PARAMETERS MEASURED: growth rate and yield at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test, temperature of the medium continuously in a temperature control vessel, appearance of the cells at the end of the final test by microscopic observations.
- 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 immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 % of a saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: no
FIRST RANGE-FINDING STUDY:
- Test concentrations: 0.1, 1.0, 10 and 100 % of a saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: no
SECOND RANGE-FINDING STUDY:
- Test concentrations: 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Two full tests were performed before the definitive test. Because results of the first full test were not in line with the results from the range-finding tests, a second full study was perfomed. Since in the second test no NOEC and EC50 values could be determined, the full test was repeated. This third full test was reported as the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (February 2017)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.4 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Details on results:
- The EC50 for growth rate inhibition (72h-ERC50) was 9.5 μg/L with a 95% confidence interval ranging from 8.7 to 10 μg/L; The 72h-NOEC for both, growth rate inhibition and yield inhibition was 0.32 μg/L; The EC10 for growth rate inhibition (72h-ERC10) was 2 μg/L with a 95% confidence interval ranging from 1.6 to 2.3 μg/L
- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.

The measured concentrations in nominally 40, 200 and 1000 μg/L were at a level of 79-89% of nominal at the start of the exposure. In the two lowest groups the measured concentrations were below the lowest calibration standard from the start of the exposure. The measured concentration in nominally 40 μg/L decreased below the lowest calibration standard already after 24 hours of exposure while measured concentrations in the two highest groups were at the level of 20 and 58% of the initial at the end of the test, respectively.
As actual exposure concentrations could not be reliably calculated based on the analytical results, it was decided to express the effect parameters in terms of nominal concentrations. A justification for using nominal concentrations was given in the 'Overall remarks' section.

Individual pH and temperature values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.2 mg/L (95% CI: 1.1-1.2 mg/L)
- Other: results fell within the historical range.
Reported statistics and error estimates:
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 (Williams Multiple Sequential t-test Procedure) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm, both 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 in OECD 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 the test item.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1 Measured concentrations in the definitive test.

Time of sampling
[hours]

Concentration
[µg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analysed

 

 

 

 

 

0

0

< 4.00*

n.a.

 

 

0.32

< 4.00*

n.a.

 

 

1.6

< 4.00*

n.a.

 

 

8.0

12.3

154

 

 

8.0**

12.4

155

 

 

40

35.1

88

 

 

200

157

79

 

 

1000

892

89

 

 

 

 

 

 

 

 

 

 

 

24

0

n.d.

n.a.

n.a.

 

0.32

n.d.

n.a.

n.a.

 

1.6

< 4.00*

n.a.

n.a.

 

8.0

< 4.00*

n.a.

n.a.

 

8.0**

< 4.00*

n.a.

n.a.

 

40

< 4.00*

n.a.

n.a.

 

200

57.4

29

36

 

1000

667

67

75

 

 

 

 

 

 

 

 

 

 

72

0

< 4.00*

n.a.

n.a.

 

0.32

< 4.00*

n.a.

n.a.

 

1.6

< 4.00*

n.a.

n.a.

 

8.0

< 4.00*

n.a.

n.a.

 

8.0**

< 4.00*

n.a.

n.a.

 

40

< 4.00*

n.a.

n.a.

 

200

31.1

16

20

 

1000

514

51

58

*     The value could not be estimated correctly, therefore it is reported as lower than the lowest acceptable calibration standard.

**   Without algae.

n.d.       Not detected.; n.a.       Not applicable.

Table 2       Growth rate and percentage inhibition for the total test period during the definitive test

Test item

Nominal concentration (µg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.567

0.0424

6

0.32

1.511

0.0930

3

3.6

1.6

1.307*

0.0362

3

17

8.0

0.979*

0.0484

3

38

40

0.063*

0.0580

3

96

200

0.113*

0.1032

3

93

1000

0.000*

0.0000

3

100

* - effect statistically significant when compared to the control (α<0.05)

Table 3        Growth rate and percentage inhibition at different time intervals during the definitive test

Test item

Nominal conc. (µg/L) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.281

 

1.559

 

1.861

 

0.32

3

0.836

35

1.864

-20

1.834

1.5

1.6

3

0.000

100

1.897

-22

2.026

-8.9

8.0

3

0.467

64

0.261

83

2.211

-19

40

3

0.050

96

-0.050

103

0.188

90

200

3

0.010

99

-0.010

101

0.339

82

1000

3

0.084

94

-0.084

105

0.000

100

Acceptability of the test:

1). In the control, cell density increased by an average factor of > 16 within two days (i.e. 111).

2). The mean coefficient of variability for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 21%).

3). The coefficient of variation of average specific grwoth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2.7%).

All validity criteria were met, therefore the study was considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'any other information on results' section.
Conclusions:
The EC50 for growth rate inhibition (72h-ERC50) was 9.5 µg/L with a 95% confidence interval ranging from 8.7 to 10 µg/L. The 72h-NOEC for both, growth rate inhibition and yield inhibition was 0.32 µg/L. The EC10 for growth rate inhibition (72h-ERC10) was 2 μg/L with a 95% confidence interval ranging from 1.6 to 2.3 μg/L
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at the following nominal concentrations 0 (blank-control), 0.32, 1.6, 8.0, 8.0 (without algae), 40, 200 and 1000 µg/L. Exposure concentrations were verified to be 79 -89% of nominal at the start of the exposure for 40, 200 and 1000 µg/L. Exposure concentrations were 20 and 58% for 200 and 1000 µg/L, respectively. For other concentrations, no values could be measured at different timed during the test because concentrations decreased below the lowest calibration standard. As actual exposure concentrations could not be reliably calculated based on the analytical results, effect parameters were expressed as nominal concentrations. The EC50 for growth rate reduction (72h-ERC50) was 9.5 µg/L (95% confidence levels 8.7 - 10 µg/L) and the 72h-NOEC for growth rate reduction was 0.32 µg/L. The study is considered to be reliable without resitrictions.