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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Dec 2017 - 02 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test item information:
Identification: Gurjun Balsam Oil (Gurjunene)
Appearance: Pale yellow oily liquid
Batch: 2642704
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 28 February 2018 (expiry date)

Additional information:
Test Facility test item number: 209144/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Essential oil obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)
Specific gravity / density: 0.9171 (20/20°C)

Analytical monitoring:
yes
Remarks:
TOC measurement
Details on sampling:
The extra replicates without algae and without HEPES were used for possible analysis 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.

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.
Vehicle:
no
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of Gurjun Balsam Oil (Gurjunene) tested was a pale yellow oily liquid. The test item was a UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. During preparation of test solutions the test items were treated as being possibly volatile. Preparation of test solutions started with loading rates individually prepared at concentrations ranging from 0.10 to 100 mg/L. A two-day period of magnetic stirring in closed vessels with minimal headspace in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained aqueous mixtures were allowed to settle overnight. Thereafter, the Water Accommodated Fractions (WAFs) were collected by means of siphoning and microscopically inspected for the presence of undissolved test material. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 40 mL were added to each replicate vessel of the respective test concentration containing a 0.8 mL of an algal suspension providing a cell density of 10^4 cells/mL. Any residual volumes were discarded.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.
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 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator varied between 22 and 23°C.
pH:
t=0h : 7.2 - 7.3
t =72h: 7.4 - 7.9
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: WAFs prepared at loading rates of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

TOC measurements:
Loading rates: Control, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
TOC t=0h : n.a, n.q, n.q, n.q, n.q, n.q, 1.1 and 5.8 mg/L
TOC t=72h: n.a, n.q, n.q, n.q, n.q, 0.17, 1.3 and 6.1 mg/L

n.a. – not applicable, n.q. – not quantifiable

Details on test conditions:
TEST SYSTEM
- Gurjun Balsam Oil (Gurjunene): WAFs at 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Controls: Test medium without test item or other additives.
- Replicates
- 3 replicates of each WAF
- 6 replicates of the control
- 2 extra replicates of each test group without algae and without HEPES for TOC analysis
- 1 extra replicate of each WAF without algae as a possible background for treated solutions
- Test duration: 72 hours
- Test type: Static
- Test vessels: 40 mL, all-glass, closed with no headspace
- Medium: Adjusted M2
- Cell density: An initial cell density of 1 x 10^4 cells/mL.

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 72 to 75 µE.m-2.s-1.
- Incubation: Airtight closed vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENT AND RECORDINGS
- 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 WAF at 32 mg/L and the control to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 cuvettes (path length =10 mm). Algal medium was used as blank.

RANGE- FINDING TEST:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at 1.0, 10 and 100 mg/L and to a control
• Cell densities were recorded only at the end of the exposure period
• One extra test vessel per concentration without algae was used as background for the determination of the algal cell density
• pH was only measured in the control and the WAF at 100 mg/L
• At the end of the test algae were not observed to verify a normal and healthy appearance.

- Results used to determine the conditions for the definitive study: Yes. The expected EL50 for growth rate inhibition was above a WAF prepared at a loading rate of 100 mg/L. The expected EL50 for yield inhibition was between WAFs prepared at loading rates of 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
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:
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
8.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 5.7 - 11
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance,
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 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.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 10 - 17
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.11-0.36
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Measured Test Item Concentrations:
TOC concentrations in the test solutions proved to be too low to measure reliably (almost all <1 mg/L), but were in line with the results from the range finding test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Inhibition of Growth Rate and Inhibition of Yield:
Inhibition of growth rate increased with increasing loading rates of Gurjun Balsam Oil (Gurjunene) resulting in 26% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at loading rates of 0.32 mg/L and higher. However, only the inhibition found at loading rates of 32 mg/L and higher was found to be biologically relevant, i.e. was >10%. Inhibition of yield increased with increasing loading rates of Gurjun Balsam Oil (Gurjunene) resulting in 77% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at loading rates of 0.32 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF at 32 mg/L when compared to the control.

Acceptability of the Test:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 314).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 35%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1%).
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOELR 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 loading rates compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of 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 maximum 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).

Growth Rate (µ, day-1) and Percentage Inhibition for the Total Test Period

Gurjun Balsam Oil (Gurjunene),

loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.915

0.0274

6

0.10

1.884

0.0365

3

1.6

0.32

1.853

0.0083

3

3.3#

1.0

1.825

0.0432

3

4.7#

3.2

1.815

0.0365

3

5.2#

10

1.737

0.0562

3

9.3#

32

1.572

0.0599

3

17.9*

100

1.421

0.0644

3

25.8*

* Effect was statistically significant

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

Yield (x104Cells/mL) and Percentage Inhibition for the Total Test Period

Gurjun Balsam Oil (Gurjunene),

loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

312.8

25.35

6

0.10

285.1

31.42

3

8.9

0.32

258.3

6.38

3

17.4*

1.0

238.9

29.88

3

23.6*

3.2

231.5

25.18

3

26.0*

10

184.1

29.65

3

41.2*

32

111.8

19.62

3

64.3*

100

71.0

14.39

3

77.3*

* Effect was statistically significant

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, Gurjun Balsam Oil (Gurjunene) reduced growth rate of this fresh water algae species significantly at a loading rate of 32 mg/L and higher (based on biological relevance). The 72h -NOELR, ERL10 and ERL50 was 0.10 mg/L (when based on statistical analysis and 10 mg/L when based on biological relevance), 8.3 and >100 mg/L respectively.

Executive summary:

A full OECDTG 201 GLP test was performed with Pseudokirchneriella subcapitata based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg Gurjun Balsam Oil (Gurjunene) per litre under static conditions. The initial algal cell density was 10^4 cells/mL. 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. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum. Effect parameters were reported in terms of loading rates initially prepared. Inhibition of growth rate increased (slowly) with increasing loading rates resulting in 26% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at loading rates of 0.32 mg/L and higher. However, only the inhibition found at loading rates of 32 mg/L and higher was found to be biologically relevant, i.e. was >10%. Inhibition of yield increased with increasing loading rates resulting in 77% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at loading rates of 0.32 mg/L and higher.The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, the ERL50, ERL10 and 72h -NOELR for growth rate for Gurjun Balsam Oil (Gurjunene) was > 100, 8.3 and 0.10 mg/L (when based on statistical analysis and 10 mg/L when based on biological relevance). While the EYL50, EYL10 and 72h -NOELR for yield inhibition was determined as 13, 0. 22 and 0.10 mg/L respectively.

Description of key information

A full OECDTG 201 GLP test was performed with Pseudokirchneriella subcapitata based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg Gurjun Balsam Oil (Gurjunene) per litre under static conditions. The initial algal cell density was 10^4 cells/mL. 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. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum. Effect parameters were reported in terms of loading rates initially prepared. Inhibition of growth rate increased (slowly) with increasing loading rates resulting in 26% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at loading rates of 0.32 mg/L and higher. However, only the inhibition found at loading rates of 32 mg/L and higher was found to be biologically relevant, i.e. was >10%. Inhibition of yield increased with increasing loading rates resulting in 77% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at loading rates of 0.32 mg/L and higher.The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, under the conditions of this study with Pseudokirchneriella subcapitata, the ERL50, ERL10 and 72h -NOELR for growth rate for Gurjun Balsam Oil (Gurjunene) was > 100, 8.3 and 0.10 mg/L (when based on statistical analysis and 10 mg/L when based on biological relevance). While the EYL50, EYL10 and 72h -NOELR for yield inhibition was determined as 13, 0. 22 and 0.10 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
8.3 mg/L

Additional information

Effect parameters are loading (ELx) results. The ErL50 exceeds 100 mg/L WAF loading.