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:
2017
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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Remarks:
light yellow
Details on test material:
CH03216 / E1A

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h and t=72 h
Volume 3.0 mL
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

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.
Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of 3,4,5-TRIMETHOXYCINNAMIC ACID tested was a light yellow crystalline solid with a purity of approximately 99% which was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Weighing and preparation of test solutions was performed under dimmed light.
Preparation of test solutions started with the highest concentration of 100 mg/L applying a 10‑minute treatment with ultrasonic waves followed by approximately 40 minutes of magnetic stirring to accelerate dissolution of 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.
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 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 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.

ACCLIMATION
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 104 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:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
between 22 and 23°C
pH:
6.6-8.3
Nominal and measured concentrations:
1.0. 3.2, 10, 32 and 100 mg/L.
Samples taken from all test concentrations were analysed. The actual exposure concentrations were at the level of nominal throughout the exposure period (i.e., 82-101%). Based on these results, effects parameters were based on analytically confirmed nominal concentrations.

Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Static
- aeration: continuous
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 281.9 x 10^4 cells/mL
- No. of organisms per vessel: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 1 x 10^4 cells/mL
- No. of vessels per control (replicates): 1 x 10^4 cells/mL
- No. of vessels per vehicle control (replicates): 1 x 10^4 cells/mL

GROWTH MEDIUM
- Standard medium used: yes
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

TEST MEDIUM / WATER PARAMETERS
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous (24h)
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
pH At the beginning and at the end of the test.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
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 immersion probe (pathlength =20 mm for the combined limit/range-finding test; pathlength=10 mm for the final test). Algal medium was used as blank.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L.
- Results used to determine the conditions for the definitive study: yes, 1.0, 3.2, 10, 32 and 100 mg/L in the final test
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
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:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
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.
The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 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 3,4,5-TRIMETHOXYCINNAMIC ACID reduced growth rate and inhibited the yield of this fresh water algae species significantly at an analytically confirmed nominal concentration of 100 mg/L.
The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were beyond the range tested.
The 72h-NOEC for growth rate inhibition and yield inhibition was 32 mg/L.
Executive summary:

The objectiveofthe study was to evaluate 3,4,5-TRIMETHOXYCINNAMIC ACID 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 inhibition of both growth rate and 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 3,4,5-TRIMETHOXYCINNAMIC ACID tested was a light yellow crystalline solid with a purity of approximately 99% which was completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal 1.0. 3.2, 10, 32 and 100 mg/L of test item. The initial algal cell density was 104cells/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.

Both growth rates and yield were stimulated when exposed to concentrations ≤ 32 mg/L. At the highest test concentration, growth rate and yield were inhibited by 10 and 44%, respectively, and effects were statistically significant.

Samples taken from all test concentrations were analysed. The actual exposure concentrations were at the level of nominal throughout the exposure period (i.e., 82-101%). Based on these results, effect parameters were based on analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

32

>100*

>100

>100

lower 95%-CI

 

 

 

 

upper 95%-CI

 

 

 

 

Yield

Value

32

11

40

>100

lower 95%-CI

 

5.7

24

 

upper 95%-CI

 

18

76

 

* Result obtained by extrapolation was 130 mg/L (95% confidence interval ranging from 66-396 mg/L)