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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:
27 November 2018 - 30 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): 1,4-dioxan-2-one (PDO monomer)
- Physical state: solid (powder)
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: YRMC196
- Expiration date of the lot/batch:24 January 2019
- Purity test date: 12 September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator, 2-8°C
- Solubility and stability of the test substance in the solvent/vehicle: completely soluble in test medium at the concentrations tested


Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
Frequency at t=0 h and t=24 h.
Volume 4.0 mL from the approximate centre of the test vessels.
Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

At the end of the exposure period, no samples were collected and analysed for determination of exposure concentration since no test item was detected at any of the test concentrations after 24 hours of exposure.


Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve 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.
- Controls: yes

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Method of cultivation: Algae stock cultures are started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Medium M1 was used.
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture are inoculated in M2 medium at a cell density of 1 x 104 cells/mL. The pre-culture is maintained under the same conditions as used in the test. The cell density is measured immediately before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 2 to 4 days before start of the test)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
20-23°C
pH:
t=0h: 7.25
t=72h: 7.65
Dissolved oxygen:
not applicable
Salinity:
not relevant
Conductivity:
not relevant
Nominal and measured concentrations:
Nominal concentrations: 0.1, 1.0, 10, 100 mg/L
Measured concentrations (t=0h): *, 0.39, 5.4, 56 mg/L
Measured concentrations (t=24h): *

* no test item detected
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass flask
- Type: closed
- Material, size, headspace, fill volume: 40 mL airtight closed with no headspace to prevent any loss of the test item due to volatilization
- Aeration: no
- Initial cells density: 10 000 cells/ml
- No. of vessels per concentration (replicates): 6 replicates each for the limit concentration, 3 replicates each for the lower test concentration, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure
- No. of vessels per control (replicates): 6 replicates each for the control, 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505, formulated using Milli-RO water and with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium: yes (M1 versus M2)
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test in at least one vessel per concentration. Temperature was measure continuously in a temperature control vessel. At the end of the final test microscopic observation was perflormed on at least one of the test concentrations with sufficient algal growth to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: light intensity within the range of 60 to 120 μE.m-2.s-1 when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 cuvettes (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Reported statistics and error estimates:
For determination of the NOEC the approaches recommended in the OECD guideline 201 were used. 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 or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

The ECx-values could not be determined because the observed effects were below 10%.

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate and yield was recorded at the maximum concentration of 1,4-dioxan-2-one (PDO monomer) tested.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the maximum concentration tested, i.e. exceeded a nominal concentration of 100 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was beyond the maximum concentration tested, i.e. exceeded a nominal concentration of 100 mg/L.
The 72h-NOEC for growth rate inhibition was lower than a nominal concentration of 100 mg/L based on statistical significance and was set at a nominal concentration of 100 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was lower than a nominal concentration of 100 mg/L based on statistical significance and was set at a nominal concentration of 100 mg/L based on biological relevance.