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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
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Balsams, gurjun
EC Number:
232-444-8
EC Name:
Balsams, gurjun
Cas Number:
8030-55-5
IUPAC Name:
Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Copaene quality)
Test material form:
liquid
Remarks:
Clear colourless to pale yellow liquid
Details on test material:
- Name of test material (as cited in study report): Gurjun Balsam oil
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information
Specific details on test material used for the study:
Identification: GURJUN BALSAM OIL
Appearance: Clear colourless to pale yellow liquid
Batch: L4285828
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 30 April 2018 (retest date)

Test item: 207786/A
Purity/composition correction factor: No correction factor required
CAS Number: 8030-55-5 (93165-18-5)
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:
yes
Remarks:
TOC measurement
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=72 h
Volume: 40 mL
Storage: Samples were stored in a refrigerator (2-8°C) until analysis.
At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.
Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator (2-8°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
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of GURJUN BALSAM OIL tested was a clear colourless to pale yellow liquid. The item was a UVCB substance 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 individually prepared loading rates, ranging between 0.46 and 100 mg/L. Exact amounts of test substance were added to test medium containing no HEPES buffer. A 2-day period of magnetic stirring in closed vessels with minimal headspace and in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. In addition, microscopic observation of WAFs showed that they did not contain undissolved test material.

For the combined limit/range-finding test, after preparation, volumes of 110 mL were added to each replicate of the respective test concentration. Subsequently, 2.5 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.66 ml) was spiked to each vessel. For the range-finding and the final test, after preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.80 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.24 ml) was spiked to each vessel.

Solutions for analysis of TOC concentrations:
• At the start of the test: samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
• At the end of the exposure period: volumes of 110 mL (combined limit/range-finding test) or 40 mL (range-finding and full test) of each WAF were added to vessels with no algae at the start of the test and used as solutions for analysis of TOC concentrations at the end of the test. These vessels were not spiked with HEPES buffer. This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.
Any residual volumes were discarded.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an 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.
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 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:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
pH:
t=0h : 7.1 - 7.2
t =72h: 7.5 - 7.8
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).
Nominal and measured concentrations:
Nominal: 0.23, 0.46, 1.0, 2.2, 4.6, 10 mg/L
TOC measurements corrected for controls:
TOC t=0h : not quantifiable, 0.014, 0.40, not quantifiable, 1.4, 0.39 mg TOC/L
TOC t=72h: 0.52, 0.39, 0.58, 1.3, 1.1, 0.95 mg TOC/L

The TOC content of the pure substance was 84.39%
Details on test conditions:
GURJUN BALSAM OIL: WAFs prepared at loading rate of 0.23, 0.46, 1.0, 2.2, 4.6 and 10 mg/L.
Controls: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration; 6 replicates of the control; 2 extra replicates of each test concentration and the control without algae for sampling purposes; 1 extra replicate of each test item concentration without algae as a background for treated solutions.

Test duration: 72 hours
Test type: Static
Test vessels : 40 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
Medium: Adjusted M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 84 µE.m-2.s-1.
Incubation: Airtight closed vessels were distributed at random in the incubator and repositioned daily. During incubation the algal cells were kept in suspension by continuous shaking.

The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the full test with the following exceptions:
• Three replicates per concentration were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L.
• 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.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• 50 mL samples were taken for analysis

A range-finding test was performed to provide information about the range of concentrations to be used in the full test. Test procedure and conditions were similar to those applied in the full test with the following exceptions:
• Exponentially growing algal cultures were exposed WAFs prepared at loading rates of 0.46, 1.0, 10 and 100 mg/L. Three replicates were tested per concentration and three replicates in the control group.
• Glass wool was not used during siphoning of WAFs.
• pH was only measured in the control and the highest test concentration.
• Cell densities were determined only at the end of the exposure.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: ErL50 was the beyond the range tested, i.e. exceeded the loading rate of 10 mg/L of the final test, but exceeded 100 mg/L in the combined range finding test.
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: ErL50 was the beyond the range tested, i.e. exceeded the loading rate of 10 mg/L of the final test, but exceeded 100 mg/L in the combined range finding test.
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.3-3.5
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological significance
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
4.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95%CL: 3.6 - 5.9
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.11-0.32
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 0.23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The measured TOC concentrations increased slightly with the applied loading rate indicating proper preparation of WAFs. The obtained data is scattered and indicates that the solubility of the substance is very low. Despite the fact that no undissolved test material was observed in the solutions, it is possible that the solutions were not completely homogenous.

A dose-related inhibition of growth rate was observed in this test. The observed inhibition ranged from 3.8% at the lowest group to 18% in the highest group. Inhibition of growth rate was statistically significant at all concentrations, therefore, it was concluded that the NOEL for growth inhibition based on statistical significance was below 0.23 mg/L. However, the effects observed at the three lowest concentrations tested were considered biologically not relevant (i.e. <10%). Hence, the NOEL based on biological significance was set at 1.0 mg/L. As an alternative to NOEL, the calculated EL10 value (2.9 mg/L) can be used.
Inhibition of yield increased from 19% at the lowest loading rate to 63% in the WAF prepared at 10 mg/L. The inhibition observed in the WAF prepared at 1.0 mg/L deviated from the dose-response and was lower than in the remaining groups. The reason for this deviation is unknown. Inhibition of yield was both statistically significant and biologically relevant at all concentrations, therefore, it was concluded that the NOEL for yield inhibition was below 0.23 mg/L. As the inhibition of yield showed strong dependency on the loading rate applied, it was possible to determine the EL10 and EL20 values.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a WAF prepared at a loading rate of 46 mg/L when compared to the control.


Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 0.99 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 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 0.37 mg/L with a 95% confidence interval ranging from 0.36 to 0.37 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 was just below the expected range.
Reported statistics and error estimates:
For determination of the NOEL and the EL50 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 yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rates of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Growth Rate (µ, day -1) And Percentage Inhibition For The Total Test Period

 GURJUN BALSAM OIL loading rate (mg/L)

% inhibition

Control

 

0.23

3.8#

0.46

4.6#

1.0

0.8#

2.2

10.2*

4.6

13.5*

10

17.5*

* - effect was statistically significant, # - effect was statistically significant but biologically irrelevant (<10%)

Yield (x104 Cells/mL) And Percentage Inhibition For The Total Test Period

GURJUM BALSAM OIL loading rate (mg/L)

% inhibition

Control

 

0.23

19.3*

0.46

22.7*

1.0

3.7*

2.2

43.6*

4.6

53.3*

10

62.7*

* - effect was statistically significant

 

VALIDITY CRITERIA

  • The factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 277.
  • The coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.7%.
  • The mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 15%.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See "any other information on results incl. tables"
Conclusions:
Under the conditions of the test, the 72-h ErL50 was the beyond the range tested, i.e. exceeded the loading rate of 10 mg/L of the final test, but exceeded 100 mg/L in the combined limit/range finding test. In addition, an ErL50 > 100 mg/L was also concluded in the range finder. In conclusion, in the present study with Pseudokirchneriella subcapitata, the EL50 for growth rate inhibition (72h-ERL50) was > 100 mg/L.
Executive summary:

A full OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of 0.23, 0.46, 1.0, 2.2, 4.6 and 10 mg GURJUN BALSAM OIL per litre. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. The test item appeared to be poorly soluble in test medium, nevertheless the measured TOC concentrations increased slightly with the applied loading rate indicating proper preparation of WAFs. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, GURJUN BALSAM OIL reduced growth rate of this fresh water algae species significantly at a loading rate of 2.2 mg/L and higher (based on biological relevance). The EL50 for growth rate inhibition (72h- ErL50) was beyond the range tested, i.e. exceeded a loading rate of 10 mg/L in the final test, but exceeded 100 mg/L in the combined limit/range finder test. An ErL10 was concluded at 2.9 mg/L. The EL50 for yield inhibition (72h-EYL50) was 4.5 mg/L with a 95% confidence interval ranging from 3.6 to 5.9 mg/L.