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
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-quinuclidin-3-ol
EC Number:
246-857-6
EC Name:
(R)-quinuclidin-3-ol
Cas Number:
25333-42-0
Molecular formula:
C7H13NO
IUPAC Name:
(3R)-1-azabicyclo[2.2.2]octan-3-ol
Test material form:
solid: crystalline

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control.
Frequency at t=0 h, t=24 h and t=72 h
Volume 1.0 mL
Storage 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 an intermediate test item concentration 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.

Samples taken from nominal concentrations of 10 and 100 mg/L were analysed. The actual concentrations were at the level of 95-105% of nominal during the exposure period. Therefore, the effect parameters were expressed in terms of nominal concentrations.
Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The test item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition.

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 colourless at the end of the preparation procedure.

It should be noted that pH of the highest test concentration was adjusted from 9.6 to 8.6 with 1 M/L HCl

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 10E4 cells/mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-housde laboratory culture
- Age of inoculum (at test initiation)/Method of cultivation: 3 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 10E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Standard test duration stated for the guideline
Post exposure observation period:
not applicable

Test conditions

Hardness:
not reported
Test temperature:
21 +/- C
pH:
See Table 3 in results
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
0, 0.1, 1, 10, 100 mg/l nominal
Details on test conditions:
Test vessel: 100 mL Erlenmeyer flask, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10E4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 78 to 83 µE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Controls: Test medium without test item or other additives
Replicates
6 replicates each for the control and the limit concentration.
3 replicates of each lower test concentration.
1 replicates of each concentration without algae as a background for treated solutions.
1 replicate of each test concentration for sampling purposes at 24 hour of exposure.
1 replicate of the highest concentration without algae for sampling purposes.

MEASUREMENTS
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: No microscopic observations were performed at the end of the exposure period
Reference substance (positive control):
yes
Remarks:
not run concurrently

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
Details on results:
Inhibition of growth rate was lower than 10% at all concentrations tested and therefore, considered biologically not relevant. The statistical analysis showed, that the effect observed at the highest concentration was statistically significant.
Results with reference substance (positive control):
The objective of the present reference study was to evaluate Potassium dichromate for its ability to generate toxic effects in Pseudokirchneriella subcapitata (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield (Test Facility No. 20134686).
Start of first exposure: 25 Sep 2017
Completion last exposure: 28 Sep 2017

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 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:
Statistics were performed but biological relevence was considered for interpretation of the results of this study.

Any other information on results incl. tables

Table1          
Growth Rate And Percentage Inhibition For The Total Test Period

Test Item

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.899

0.0387

6

0.10

1.835*

0.0184

3

3.4

1.0

1.885*

0.0505

3

0.74

10

1.905*

0.0435

3

-0.32

100

1.745#

0.0133

6

8.2

* - not included in statistical analysis

#- effect was statistically significant but biologically not relevant (<10%)

Table2          
Growth Rate And Percentage Inhibition At Different Time Intervals

 

Test Item

Nominal conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.188

 

1.699

 

1.811

 

100

6

1.858

15

1.723

-1.4

1.653

8.7


Table 3          
pH Levels Recorded During the Final Test


Test Item

Nominal concentration (mg/L)

pH

t=0h

t=72h

Control

8.0

8.1

0.10

8.0

8.0

1.0

8.0

8.0

10

8.5

8.0

100

8.6

7.9

                                                                                                              



Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata the EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L.

The 72h-NOEC for growth rate inhibition was <100 mg/L based on statistical significance and 100 mg/L based on biological relevance.

The EC10 for growth rate inhibition (72h-ERC10) was beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L.