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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:
The experimental start date was 03 Apr 2017, and the experimental completion date was 01 Jun 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
EC Number:
273-222-0
EC Name:
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
Cas Number:
68953-64-0
Molecular formula:
UVCB: CnHnN2 O2 (Cl)
IUPAC Name:
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
Test material form:
liquid
Specific details on test material used for the study:
Identification Quaternium-26
Appearance Clear amber to dark amber liquid
Batch 0001954899
Purity/Composition UVCB
Test item storage At room temperature
Stable under storage conditions until 22 December 2017 (retest date)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Single samples for possible analysis were taken from all test concentrations and the control.
Frequency at t=0 h and t=72 h
Volume 2.0 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, 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 an intermediate item concentration but without algae and samples for analysis were taken at the start and at the end of the test period.
During the final test, the samples were analyzed on the day of sampling and were diluted in a 1:1 (v:v) ratio with methanol and analyzed. If necessary, the samples were further diluted with 50/50 (v/v) methanol/M2-medium to obtain concentrations within the calibration range.

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture 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.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 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: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Study design

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

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22 - 23°C
pH:
8.1 ± 0.2
Nominal and measured concentrations:
Nominal: 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg/L
Initially measured: 0.018, 0.021, 0.033, 0.058, 0.10 and 0.18 mg/L
Average measured (72h): 0.0065, 0.0071, 0.0089, 0.012, 0.016 and 0.021 mg/L
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.:
0.029 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
m/z=383.5 and m/z=411.5
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
m/z=383.5 and m/z=411.5
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
m/z=383.5 and m/z=411.5
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
m/z=383.5 and m/z=411.5
Basis for effect:
growth rate
Details on results:
Growth rates were in the range of the controls at the lowest test concentration during the 72-hour test period, whereas the growth rate of algae exposed to initial concentrations at and above 0.021 mg/L (corresponding with average concentrations of 0.0071 mg/L and higher) were increasingly reduced. Growth rate reduction was statistically significant at these test concentrations. However, the effect observed at the initial concentration of 0.021 mg/L (corresponding with an average concentration of 0.0071 mg/L) was below 10% and thus not considered as biologically relevant. The NOEC was respectively set at an initial concentration of 0.021 mg/L (corresponding with average concentration of 0.0071 mg/L).
Inhibition of yield increased with increasing concentration of Quaternium-26 at all concentrations resulting in 100% inhibition at the highest test concentration. Statistically significant inhibition of yield was found at all test concentrations. The effects observed at the lowest concentration were below 10% and thus considered to be biologically not relevant. The NOEC was respectively set at initial concentration of 0.018 mg/L (corresponding with average concentration of 0.0065 mg/L).
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to an initial concentration of 0.021 mg/L (corresponding with average concentration of 0.0071 mg/L) when compared to the control.
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 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.
The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Quaternium-26 reduced growth rate of this fresh water algae species significantly at initial concentrations at and above 0.033 mg/L (corresponding with a geometric mean concentrations of 0.0089 mg/L and higher).
The 72h-NOEC for both growth rate inhibition and yield inhibition was 0.0071 mg/L based on biological relevance and geometric mean exposure concentrations.
The EC50 for growth rate inhibition (72h-ERC50) determined based on initially measured exposure concentrations was 0.029 mg/L with a 95% confidence interval ranging from 0.028 to 0.030mg/L.
The EC50 for yield inhibition (72h-EYC50) determined based on initially measured exposure concentrations was 0.022 mg/L with a 95% confidence interval ranging from 0.022 to 0.022 mg/L.
The 72h-NOEC for both growth rate inhibition and yield inhibition was 0.018 mg/L based on biological relevance and initially measured exposure concentrations.
Executive summary:

The objectiveofthe study was to evaluate Quaternium-26 for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield. The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Quaternium-26 tested was a clear amber to dark amber liquid and completely soluble in test medium at the concentrations tested. A correction was made for the purity/composition of the test item. A correction factor of 1.67 was used. Concentration/doses are expressed as mg Quaternium-26 per litre (mg Q-26/L).

A final test was performed based on the results of a preceding combined limit/range-finding and a subsequent range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg Quaternium-26 per litre. The initial algal cell density was 104 cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and after 72 hours of exposure.

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Quaternium-26 reduced growth rate of this fresh water algae species significantly at initial concentrations at and above 0.033 mg/L (corresponding with a geometric mean concentrations of 0.0089 mg/L and higher).