2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone

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:

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 guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone

EC Number:

947-565-8

Molecular formula:

C30 H42 O4

IUPAC Name:

2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone

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, t = 24 h and t=72 h
Volume 2.0 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 2.0 mL were taken from all test solutions for possible analysis. In addition, the filter containing the undissolved residue was kept for possible analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of V134434 tested was an orange-red powder with a purity of >95 mol% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A three-day period of magnetic stirring was applied after 15 minutes of ultrasonic treatment to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. 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.

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 10^4 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:

22-24°C

pH:

8.0-8.3

Nominal and measured concentrations:

WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L.

Samples taken from the limit concentration and the control were analysed. The measured concentration at the start of the test was 0.91 µg/L at the WAF prepared at a loading rate of 100 mg/L. During the exposure period, the concentration decreased below the Limit of Quantification (i.e. LOQ = 1 µg/L). Hence, the actual exposure concentration in the sample taken after 24 hours was estimated to be 0.24 µg/L by extrapolation of the calibration curve. At the end of the test, the concentration had decreased below the Limit of Detection (i.e. LOD = 0.082 µg/L). It should be noted that a small response was measured in the control at the start of the test, however was considered to be negligible. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium throughout the test.

Based on this result, effect parameters were expressed in terms of the Time Weighted Average (TWA) concentration which was calculated to be 0.22 µg/L at the limit concentration. This value should be considered as indicative. The average exposure concentration is lower than the water solubility of V134434 determined to be 0.80 µg/L at 20ºC (Charles River Den Bosch Project 20153237). For many chemicals, the solubility in test medium is lower than in pure water due to the presence of salts and high pH compared to Milli RO. Since the preparation of test solutions was based on the recommendations for poorly soluble test items as put down in OECD Guidance Document No. 23, it can be stated that testing was performed at the maximum soluble concentration of test item in test medium.

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: 274 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 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


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: A combined limit/range-finding test was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 21 and 70 mg/L when compared to the control.

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 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 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Reported statistics and error estimates:

No effects observed above maximum solubility concentrations. Therefore, no statistical analysis was possible.

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 inhibition of growth rate or reduction of yield was recorded at the Time Weighted Average (TWA) concentration of 0.22 µg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium.
The 72h-EC50 for yield inhibition (EYC50) was beyond the range of concentrations tested, i.e. exceeded the TWA concentration of 0.22 µg/L being considered as the maximum soluble concentration of test item in test medium.
The 72h-NOEC for growth rate inhibition was 0.22 µg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 0.22 µg/L based on statistical significance.
Due to the very low solubility of V134434 in water, concentration levels that might be toxic for algae could not be reached.

Executive summary:

The objective of the study was to evaluate V134434 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and 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. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of V134434 tested was an orange-red powder with a purity of >95 mol% which was not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures per group were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition, three replicates per group were exposed to WAFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the limit concentration and the control were analysed.The measured concentrationat the start of the testwas 0.91 µg/L at the WAF prepared at a loading rate of 100 mg/L. During the exposure period, the concentration decreased below the Limit of Quantification (i.e. LOQ = 1 µg/L). Hence, the actual exposure concentration in the sample taken after 24 hours was estimated to be 0.24 µg/L by extrapolation of the calibration curve. At the end of the test, the concentration had decreased below the Limit of Detection (i.e. LOD = 0.082 µg/L). Based on this result, effect parameters were expressed in terms of the Time Weighted Average (TWA) concentration which was calculated to be 0.22 µg/L at the limit concentration.

No significant differences were recorded between the values for growth rate or yield from the control and the limit concentration, i.e. TWA concentration of 0.22 µg/L.

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 (µg/L)	NOEC	EC10	EC50
Growth rate	0.22	>0.22	>0.22
Yield	0.22	>0.22	>0.22

 

Due to the very low solubility of V134434 in water, concentration levels that might be toxic for algae could not be reached.