Registration Dossier

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:
From 2017-11-20 to 2018-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 2006, corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the test media of all test concentrations and from the control at the start of the test (without algae). At the end of the test (after 72 hours), stability samples (containing algae) were taken in duplicate from all test concentrations and from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled.
Immediately after sampling, 10 mL acetonitrile was added to each 10 mL sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (at about - 20 °C). In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.
The concentrations of TRI-N-HEXYLAMINE were analytically measured in one of the duplicate samples taken from all treatments at the sampling times of 0 and 72 hours.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
As the test item is a liquid with low water solubility, the slow-stirring method was applied for preparation of a saturated test item solution. The principle of the slow-stirring procedure is that the liquid test item with a specific gravity of < 1.0, is floating on the surface of the test water during the equilibration process while the water phase is gently stirred. In this way, the formation of micro emulsions between test item and water phase, as it can occur when intensive mixing is applied, is avoided. As during slowstirring the undissolved test item is not physically mixed into the water column, it is assumed, that the water phase is containing dissolved test item only and can be used as test medium without further treatment.
293.2 μL of test item were carefully applied (pipetted) onto the surface of 2340 mL test water. This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item of 0.798 g/cm3 (at 20 °C). No auxiliary solvent or emulsifier was used.. The mixing vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers. Thereafter slow-stirring was applied for 96 hours in the closed vessel to reach a maximum concentration of dissolved test item in the test water. The stirring time was based on the results of the water solubility study where the saturation of dissolved test item was achieved after 96 hours. After this treatment the lower part of the equilibrated test medium was carefully harvested from the mixing vessel through a tap at the bottom of the vessel. The rest of the test medium with undissolved test item floating on the surface remained in the stirring vessel. The equilibrated test medium was used as stock solution, which was further used in a series of dilutions with test water to prepare the lower concentrated test media. All solutions were clear with no evidence of undissolved test item. Additionally, a control (test water only) was run in parallel.
The test media were prepared just before the start of the test. The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions were clear with no evidence of undissolved test item.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
- Method of cultivation: The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no since same cultivation and test conditions.
- Culturing media and conditions (same as test or not): yes: The algae were cultivated and tested in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 as a closed test system was applied.
- Any deformed or abnormal cells observed: no

Study design

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

Test conditions

Hardness:
The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).
Test temperature:
The water temperature during the test was maintained at 23 °C.
pH:
The pH in the control was 7.5 at test start and 7.7 at test end. The pH of the test media was in the range of 7.4 to 7.7 during the test period.
Nominal and measured concentrations:
Nominal: Dilutions of the equilibrated test medium with a loading rate of 100 mg/L: 1:800, 1:400, 1:200, 1:100, 1:50, 1:25
Geometric mean measured concentrations at both sampling dates (0 and 72 hours): 6.6, 16, 26, 58, 80, 272 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Type, Material, size, headspace, fill volume: Since the test item was determined to be volatile, glass stoppered Erlenmeyer flasks were used completely filled (without headspace) with about 60 mL of test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system)
- Aeration: no, but orbital shaker was used.
- Initial cells density: 5000 cells/mL corresponding to 0.95 x E+4 relative fluorescence units.
- Control end cells density: 230 x E+4 relative fluorescence units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes; The algae were cultivated and tested in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 as a closed test system was applied.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were dissolved in sterile purified water. The concentration of NaHCO3 was increased from 15 to 250 mg/L (as carbon source for the algal growth) and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the pH of the test media as constant as possible. The pH of the test water was adjusted to 7.5.
The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

- Intervals of water quality measurement: The light intensity was measured at the start of the test. 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 during the exposure period.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: only the pH of the test water was adjusted to 7.5.
- Photoperiod/Light intensity and quality: continuously illuminated by LED light installed above the test flasks. The light intensity at the level of the test solutions was approximately 65 μE s-1 m-2 (range: 63 to 68 μE s-1 m-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 as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A small volume (100 μL per sample) 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.
- Other: At the end of the test, a sample was taken from the control and from the dilution 1:100 to determine under the microscope a potential influence of the test item on the shape and size of the algal cells. This test concentration was chosen since the algal cell density at the two highest dilutions (1:50 and 1:25) were too low for a reliable examination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, 2 were performed:
- Test concentrations: 1) the undiluted test medium with a loading rate of 100 mg/L and the dilutions 1:5 and 1:25. 2) 1:5, 1:25, 1:125 and 1:625
- Results used to determine the conditions for the definitive study:
1) 1:25: 110% inhibition of average growth rate at 72h
1:5: 182% inhibition of average growth rate at 72h
Undiluted test medium: 206% inhibition of average growth rate at 72h
2) 1:625: -0.5% inhibition of average growth rate at 72h
1:125: 15% inhibition of average growth rate at 72h
1:25: 94% inhibition of average growth rate at 72h
1:5 : 133% inhibition of average growth rate at 72h
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
54 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 52-56 µg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
34 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 31-37 µg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
40 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 38-42 µg/L
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:100 (mean measured 58 μg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All test media were clear solutions throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate in the reference test was 1.1 mg/L (October 2017) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L).
Reported statistics and error estimates:
Growth rate and yield were calculated for each test flask. The mean values for growth rate μ and yield Y were calculated for each treatment.
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test where appropriate. Statistical analysis was performed using ToxRat Professional®.

Any other information on results incl. tables

The measured concentrations of the test item in the test media are shown in the table below. Except for the dilution 1:50, the test item concentrations did not decrease over the test period. The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at both sampling dates (0 and 72 hours).

Treatment / Dilution°

Analytically Measured Concentration
of the Test Item [µg/L]

Mean Measured Concentration (Geometric Mean)

[µg/L]

0 Hours

72 Hours

 

1:800

7.2

6.0

6.6

1:400

14

18

16

1:200

25

28

26

1:100

57

60

58

1:50

107

60

80

1:25

286

258

272

°:        Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

 

The biological results are based on the mean measured concentrations.

Growth rate:

At the mean measured concentrations of 6.6 to 272 µg/L, the mean inhibitions compared to the control were in the range of 1.0 to 112 % and were statistically significantly different from the control (results of Welch t-test, one-sided smaller, α = 0.05).

 However, the statistically significant findings at the two lowest test concentrations of 6.6 and 16 µg/L (mean inhibitions compared to the control of 1.0 and 2.3 %, respectively) were not estimated as biologically relevant toxic effects, since the variability between replicates within these concentrations and the control was very low (coefficient of variations in the range of 0.3 to 0.5 %). Moreover the mean inhibitions compared to the control were below 10 %. The EC10 for growth rate was calculated to be 34 µg/L.

Therefore, the NOEC for growth rate was determined to be at the mean measured concentration of 16 µg/L.

Yield:

At the mean measured concentrations of 6.6 to 272 µg/L, the mean inhibitions compared to the control were in the range of 5.5 to 100 % and were statistically significantly different from the control (results of Welch t-test, one-sided smaller, α = 0.05).

However, the statistically significant finding at the lowest test concentration of 6.6 µg/L (mean inhibition compared to the control of 5.5%) was not estimated as a biologically relevant toxic effect, since the variability between replicates within this concentration and the control was very low (coefficient of variation of 1.8 and 2.4 %, respectively). Moreover the mean inhibition compared to the control was below 10 %. The EC10 for this endpoint was calculated to be 15 µg/L.

Therefore, the NOEC for yield was determined to be at the mean measured concentration of 6.6 µg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the ctrl, biomass increased by 243 over 72h. The mean CV of the daily growth rates in the ctrl (section-by-section growth rates) during 72h was 13%. The CV of the average specific growth rates in the replicates of the ctrl after 72h was 0.4%.
Conclusions:
Endpoint after 72 Hours:
EC10: 34 µg/L (growth rate), 15 µg/L (yield)
EC20: 40 µg/L (growth rate), 18 µg/L (yield)
EC50: 54 µg/L (growth rate), 27 µg/L (yield)
NOEC: 16 µg/L (growth rate), 6.6 µg/L (yield)
LOEC: 26 µg/L (growth rate), 16 µg/L (yield)
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No 2016/266, C.3, and following GLP. As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled with test medium, minimizing the air space in the flasks and avoiding potential losses of test item by evaporation. As the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro-droplets) was applied for the preparation of a saturated test item solution. For the preparation of the stock solution, the test item with specific gravity of 0.798 was carefully applied (pipetted) onto the surface of the test water at a loading rate of 100 mg/L. Thereafter slow-stirring was applied for 96 hours in a closed vessel to reach a maximum concentration of dissolved test item in the test water. The stirring vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers to avoid losses of the volatile test item during stirring. After this treatment the lower part of the equilibrated test medium was carefully harvested from the stirring vessel through a tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item only, was used in a series of dilutions with test water to obtain the dilutions 1:25, 1:50, 1:100, 1:200, 1:400 and 1:800 to be tested. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Except for the dilution 1:50, the test item concentrations did not decrease over the test period. The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at both sampling dates (0 and 72 hours). The biological results are based on the mean measured test item concentrations.

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours. A statistically significant inhibitory effect on the growth rate and yield of the algae was observed at the end of the test at the mean measured concentrations of 26 and 16 μg/L, respectively. Endpoint after 72 Hours:

EC10: 34 µg/L (growth rate), 15 µg/L (yield)

EC20: 40 µg/L (growth rate), 18 µg/L (yield)

EC50: 54 µg/L (growth rate), 27 µg/L (yield)

NOEC: 16 µg/L (growth rate), 6.6 µg/L (yield)

LOEC: 26 µg/L (growth rate), 16 µg/L (yield)

All validity criteria (i.e. increase of biomass, mean coefficient of variation of the daily growth rates and coefficient of variation of the average specific growth rates) were fulfilled.