Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jul - 12 Oct 2016
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:
2006, corrected 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
No 2016/266
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Federal Office for the Environment (FOEN)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples without algae were taken were taken from the test media of all test concentrations and the control at the start and the end of the test (72 hours). For sampling at the end of the test, the test medium of the treatment replicates was pooled. Immediately after sampling, acetonitrile (10 mL acetonitrile and 10 mL sample volume) was added to each sample.
- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Since the test item was determined to be volatile, the test was performed in Erlenmeyer flasks nearly completely filled (without headspace) with 60 mL test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system) and was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours in a closed vessel. After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the undiluted filtrate were used as test media.
- Eluate: no
- Differential loading: The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the undiluted filtrate were used as test media.
- Controls: yes (test medium control)
- Evidence of undissolved material: It was assumed (and visually checked by the Tyndall effect) that the lower phase of the water column contained dissolved test item only and therefore no additional filtration was applied.
- Others: At the start of the test (Day 0) an emulsion with a loading rate of 100 mg/L was prepared by weighing 231 mg of the test item in 2310 mL test water. The test item was mixed into test water by intensive stirring for three hours in the dark in a closed stirring vessel to dissolve a maximum of test item in test water.The stirring time was based on the stirring pre-experiment which showed, that the maximum amount of dissolved test item was reached after this stirring time.After stirring, the emulsion was allowed to separate for 30 minutes and thereafter the lower phase of the water column still containing a little amount of undissolved test item was filtered through a 0.45 μm membrane filter (Whatman, NC45). As a pre-caution, the filter was preconditioned with 500 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Strain No. 61.81
- Age of inoculum: An inoculum culture was set up three days before the start of the exposure.
- Method of cultivation: The algae were cultivated at IES Laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 3 days before the start of the exposure
- Culturing media and conditions: AAP medium (according to the test guidelines), according to the closed system NaHCO3 was increased by 200 mg/L to 250 mg/L and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
23 °C (through the whole test period)
pH:
7.9 (0 h test treatment) 7.9 - 8.0 (72 h test treatment)
7.9 (0h control) 8.0 (control test end 72h)
Nominal and measured concentrations:
nominal: control, dilutions 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate (undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L)
geometric mean measured concentration: 0.10, 0.31, 1.1, 3.2 and 11 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks nearly filled completly with 60 mL (without headspace) test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system)
- Aeration: No, constantly shaken (orbital shaker, Multitron-Pro, Infors HT, Bottmingen/Switzerland). The test flasks were positioned randomly and repositioned daily.
- Initial cells density: The test was started using a nominal algal cell density of 5000 cells/mL (corresponding to 0.66 x 10^4 cells per mL).
- Control end cells density: 104.5 x 10^4 relative fluorescence units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium, prepared according to OECD but modified due to closed test system according to the International Standard ISO 14442
- Detailed composition if non-standard medium was used: NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: HEPES-buffer were added to keep the pH of the test media as constant as possible
- Photoperiod: 72 h
- Light intensity and quality: approximately 65 μE s-1m-2 (range: 62 to 68 μE s-1m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A small volume (2 x 100 μL) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- Chlorophyll measurement: fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK)
- Microscopic evaluation: At the end of the test showed no difference between the algae growing at the test concentration of 1.1 mg/L (dilution 1:10) and the algal cells in the control was observed. The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: control, 1:20, 1:5 and undiluted filtrate (undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L)
- Results used to determine the conditions for the definitive study: Dilution 1:20: 6% inhibition of growth, Dilution 1:5 : 29% inhibition of growth, Undiluted filtrate: 155% inhibition of growth
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-CI:
Remarks:
1.0 - 1.4 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-CI:
Remarks:
2.0 - 2.4 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-CI:
Remarks:
0.74 - 0.88 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-CI:
Remarks:
1.3 - 1.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- Unusual cell shape: The shape and size of the algal cells were obviously not affected by the test item up to at least 1.1 mg/L (dilution 1:10) and the algal cells in the control.
- Any stimulation of growth found in any treatment: yes, at 0.10 mg/L (dilution 1:100) an inhibition of -1.1 % was determined after 72 h
- Any observations that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- Other: Analytical and biological results are summarized within the tables 1-4 in the section "Any other information on results incl. tables"
Results with reference substance (positive control):
- EC50: 72-hour was 1.0 mg/L (within the range recommended by the guideline: 72-hour ErC50: 0.92-1.46)
Reported statistics and error estimates:
The 72-hour EC10 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression. For the determination of the NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test, or Welch t-test where appropriate. Statistical analysis was performed using ToxRat Professional®. Based on the ToxRat evaluation of the dat set Welch t-test was used, one-sided smaller, α = 0.05.

At the end of the test, the test item concentrations were in the range of 67 to 99% of the initially measured values. Due to the slight decrease of test item concentrations during the test period of 72 hours, the mean measured test item concentrations over the test period were calculated as the geometric mean of the test item concentrations measured at the start and the end of the test.

Table 1: Analytical measured concentration of the test item  

Treatment

Dilution

Analytical Measured Concentration of the Test Item [mg/L]

Mean Measured Concentration (Geometric Mean)

[mg/L]

0 hours

72 hours

Dilution 1:100

0.121

0.0808

0.10

Dilution 1:32

0.327

0.295

0.31

Dilution 1:10

1.15

1.13

1.1

Dilution 1:3.2

3.36

2.98

3.2

Undiluted Filtrate°

11.3

11.2

11

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L

Table 2: Analytical results of the test item  

Sampling Day /Age of Sample

Dilution Factor of Filtrate of Loading Rate 100 mg Test Item /L

Measured Concentration of Test Item

x

Sample Preparation Factor

F

Determined Concentration of Test Item

c

% of Initially Measured Concentration

[d/h]

 

[mg/L]

 

[mg/L]

[%]

0/0

(fresh)

Control

< 0.05

2

< LOQ

 

1:100

0.0605

2

0.121

 

1:32

0.164

2

0.327

 

1:10

0.574

2

1.15

 

1:3.2

1.68

2

3.36

 

Undiluted Filtrate

5.64

2

11.3

 

3/72

(aged)

Control

< 0.03

2

< LOQ

n.a.

1:100

0.0404

2

0.0808

67

1:32

0.147

2

0.295

90

1:10

0.566

2

1.13

99

1:3.2

1.49

2

2.98

89

Undiluted Filtrate

5.62

2

11.2

99

 Table 3: Results of Growth rate and Growth rate inhibition

Treatment Dilution

Mean Measured Concentration

Average Growth Rate μ (day-1) and Inhibition of μ (Ir)

0-24 h

0-48 h

0-72 h

[mg/L]

μ

Ir [%]

μ

Ir [%]

μ

Ir [%]

Control

---

1.722

0.0

1.781

0.0

1.689

0.0

1:100

0.10

1.657

3.8

1.752

1.6

1.707

-1.1

1:32

0.31

1.767

-2.6

1.750

1.8

1.679

0.6

1:10

1.1

1.596

7.3

1.647#

7.5

1.573#

6.9

1:3.2

3.2

0.893*

48.2

0.591#

66.8

0.350#

79.3

Undilut. Filtrate°

11

-0.989*

157.4

-0.875#

149.1

-0.779#

146.1

 Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

#: Mean value statistically significantly lower than in the control (according toWelch t-test, one-sided smaller, α = 0.05)

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 Table 4: Results of Yield and Yield inhibition

Treatment Dilution

Mean Measured Concentration

Yield Y (x 104) and Inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

[mg/L]

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

---

3.0

0.0

22.5

0.0

103.9

0.0

1:100

0.10

2.8

6.7

21.2

5.9

109.5

-5.4

1:32

0.31

3.2

-5.7

21.2

6.0

100.7

3.1

1:10

1.1

2.6

14.0

17.1*

24.1

73.0#

29.7

1:3.2

3.2

1.0#

68.1

1.6*

93.0

1.2#

98.8

Undilut. Filtrate°

11

-0.4#

113.6

-0.5*

102.4

-0.6#

100.6

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller,α= 0.05).

#: Mean value statistically significantly lower than in the control (according toWelch t-test, one-sided

smaller, α = 0.05)

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Validity criteria fulfilled:
yes
Conclusions:
The test item had toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a 72-hour static test according to OECD 201 and GLP.
The algae study resulted in an ErC50 (72 h) of 2.2 mg/L and an ErC10 (72 h) of 1.2 mg/L (based on the geometric mean measured concentrations).

Description of key information

ErC50 (72 h) = 2.2 mg/L (Pseudokirchneriella subcapitata, OECD 201, based on geometric mean measured concentration)

ErC10 (72 h) = 1.2 mg/L (Pseudokirchneriella subcapitata; OECD 201, based on geometric mean measured concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:
2.2 mg/L
EC10 or NOEC for freshwater algae:
1.2 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata.

As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). Due to the low water solubility of the test item, the test media were prepared following the filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours in a closed vessel. After stirring the emulsion was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the undiluted filtrate were used as test media.

At the end of the test, the test item concentrations were in the range of 67 to 99 % of the initially measured values (slight decrease of test item concentrations during the test period of 72 hours).

Inhibition of growth was recorded resulting in an ErC10 (72 h) of 1.2 mg/L and an ErC50 (72 h) 2.2 mg/L (based on the geometric mean measured concentrations).