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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 May 2018 - 17 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006 (Annex 5 corrected 28 July 2011)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
6-hexyltetrahydro-2H-pyran-2-one
EC Number:
211-915-1
EC Name:
6-hexyltetrahydro-2H-pyran-2-one
Cas Number:
710-04-3
Molecular formula:
C11H20O2
IUPAC Name:
6-hexyloxan-2-one
Test material form:
liquid
Details on test material:
Appearance: Clear colourless to pale yellow liquid
Test item storage: In refrigerator (2-8°C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations.
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: 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 10 mg/L 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium. Test solutions were prepared at the highest concentration of 100 mg/L applying an overnight period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration 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.
- Controls: Test medium without test item or other additives.

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
- Age of inoculum (at test initiation): 4 days
- 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.
- Pre-culture: 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.
- Stock culture medium: M1, according to NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505).
- Pre-culture medium and test medium: M2, according to OECD 201.

Study design

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

Test conditions

Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21 - 23 °C
pH:
At t=0 h: 8.0-8.2
At t=72 h: 8.0-8.2
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32, 100 mg/L
Measured concentrations were 90-99% of nominal at the start of the test and decread to 21-42% of nominal at the end of the test. Based on these results, the Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters. See Table 1 in 'Any other information on results incl. tables' for details on measured concentrations throughout the test and Table 2 for TWA concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean): 112.3 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure
- 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium, formulated using Milli-RO water (tap-water purified by reverse osmosis).
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 72 to 76 μE/m^2/s

EFFECT PARAMETERS MEASURED
- Cell density was measured at t= 24, 48, and 72 h.
- Determination of cell concentrations: During the final test particles were observed at the highest test concentration at 72 hours of exposure. These particles disturbed spectrophotometric measurement. Therefore, algal density was determined by use of a microscope and a counting chamber throughout the study.

COMBINED LIMIT/RANGE-FINDING TEST
- Concentrations: Control, 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected ErC50 was between 10 and 100 mg/L (nominal concentration).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed July 2018)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
27 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% c.i.: 25-29 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% c.i.: 3.7-4.8 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
meas. (TWA)
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.:
1.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of all the algal cells exposed to the test item when compared to the control.
- Inhibition of growth rate increased with increasing concentrations of test item from 0.50 mg/L upwards resulting in 77 inhibition at 67 mg/L. Statistically significant inhibition of growth rate was found at a TWA concentration of 1.7 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 1.7 mg/L, at which the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 1.7 mg/L for growth rate inhibition.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- In the chromatogram of the nominal 100 mg/L sample analysed at the end of the combined limit/range-finding test, an additional peak was observed. The additional peak probably derives from a degradation product of the test item but was not observed during the final test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
ECx: Probit analysis using linear max. likelihood regression.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Final Test Test: Test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis *

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

03 Jul 2018

17 Jul 2018

0

n.d.

n.a.

 

 

 

 

1.0

0.898

90

 

 

 

 

3.2

3.15

99

 

 

 

 

10

9.78

98

 

 

 

 

10#

9.72

97

 

 

 

 

32

28.8

90

 

 

 

 

100

92.2

92

 

24

04 Jul 2018

17 Jul 2018

0

n.d.

n.a.

n.a.

 

 

 

1.0

0.641

64

71

 

 

 

3.2

2.15

67

68

 

 

 

10

7.13

71

73

 

 

 

10#

7.76

78

80

 

 

 

32

23.5

74

82

 

 

 

100

79.0

79

86

48

06 Jul 2018

17 Jul 2018

0

n.d.

n.a.

n.a.

 

 

 

1.0

0.216

22

24

 

 

 

3.2

0.658

21

21

 

 

 

10

2.79

28

29

 

 

 

10#

3.81

38

39

 

 

 

32

9.23

29

32

 

 

 

100

42.5

42

46

*     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

#     Without algae.

n.d.Not detected.

n.a.Not applicable

Table 2: Time Weighted Average Versus Nominal Concentrations

Test item

Nominal conc. (mg/L)

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72 h

1.0

0.90

0.64

0.22

0.50

3.2

3.2

2.2

0.66

1.7

10

9.8

7.1

2.8

5.8

32

29

24

9.2

18

100

92

79

42

67

Table 3: Growth Rate and Percentage Inhibition for the Total Test Period

Test item
TWA conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.568

0.0668

6

 

0.50

1.549

0.0208

3

1.2

1.7

1.464

0.0119

3

 6.7#

5.8

1.300

0.0294

3

 17*

18

1.030

0.0500

3

 34*

67

0.363

0.0561

3

 77*

* - effect was statistically significant,#- effect was statistically significant however biologically not relevant (<10%).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria
Conclusions:
The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 27 mg/L, 4.2 mg/L and 1.7 mg/L (based on biological relevance), based on Time Weighted Average concentrations.
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 nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (triplicate) and an untreated control (6 replicates). Measured concentrations in the test solution were at 90 - 99% relative to nominal at the start of the test and decreased to 21 - 42% at the end of the test. Based on these results the Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters. TWA concentrations were 0.50, 1.7, 5.8, 18 and 67 mg/L. The EC50 for growth rate inhibition (72h-ErC50) was 27 mg/L, the 72h-ErC10 was 4.2 mg/L. The 72h-NOErC was 0.50 and 1.7 mg/L based on statistical significance and biological relevance, respectively. The study met all validity criteria, and is considered reliable without restriction.