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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:
26 May 2017 - 13 July 2017
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:
28 July 2011
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
OECD series on testing and assessment number 23, 2000
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Copaiba balsam oil
Appearance: Pale yellow liquid
Batch: S-71699
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 14 February 2019 (expiry date)
Test Facility test item number: 208338/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Essential oil of Copaiba balsam obtained fromthe exudate of the Copaifera tree by distillation
Molecular structure: UVCB
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available
Analytical monitoring:
yes
Remarks:
TOC analysis to confirm WAF preparation
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.

Vehicle:
no
Remarks:
Water Accomodated Fractions were prepared
Details on test solutions:
The batch of Copaiba balsam oil tested was a pale yellow liquid. The test item was a UVCB substance poorly 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 ranging between 1.0 and 100 mg/L. Exact amounts of test item 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. Final test solutions were observed to be hazy at WAFs prepared at loading rate of 10 mg/L and higher, while all the remaining test solutions were clear and colorless. Microscopic observation of the WAFs showed that they did not contain undissolved test material.

After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 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 40 mL of each WAFs 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 (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- 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. 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.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Remarks:
adjusted M2
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. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
pH:
Start of the experiment: (0h) : 7.1 - 7.2
End of the experiment (72h): 7.4 - 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: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
TOC measurements:
TOC t=0h : n.q, n.q, 0.82, 1.3, 5.4 mg TOC/L
TOC t=72h: n.q., n.q, n.q, 1.3, 3.7 mg TOC/L

n.q.: not quantifiable
n.d.: not determined
Details on test conditions:
TEST CONCENTRATIONS
Copaiba balsam oil: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 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 and HEPES for sampling purposes;
- 1 extra replicate of each test item concentration without algae as a background for treated solutions.

TEST PROCEDURE AND CONDITIONS
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 10^ 4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 85 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.

MEASUREMENTS 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 the control and the highest concentration tested to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED
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 and the extra replicates as a background for treated solutions.

TEST CONCENTRATIONS

Spacing factor for test concentrations:3.2

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:
• Exponentially growing algal cultures were exposed to WAFs prepared at loading rates of 1.0 to 100 mg/L increasing by a factor of 10 and to a control.
• Three replicates were tested per loading rate and three replicates in the control group.
• pH was only measured in the control and the highest test loading rate.
• 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.

Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Copaiba balsam oil proved to be very poorly soluble in test medium as only 5.1-10% of the expected TOC concentration was measured at the start of the test. Nevertheless, the measured TOC concentrations increased with the applied loading rate indicating proper preparation of WAFs. It should be noted, that values below 1.0 mg TOC/L should be considered indicative. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. Statistical significant inhibition of growth rate and yield was found at all loading rates. A flat dose-response curve was observed; i.e. growth rate was inhibited by 28% at WAF prepared at loading rate of 1.0 mg/L and by 40% at WAF prepared at a loading rate of 100 mg/L. For this reason, no ELx (x=10, 20 and 50) or NOEL could be calculated. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to WAF prepared at loading rate of 100 mg/L when compared to the control.

Experimental conditions:
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator varied between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Validity criteria:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 197).

2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 21%).

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. 1.7%).

Results with reference substance (positive control):
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 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 inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). No ELx-values (x=10, 20 and 50) could be calculated since they were below 50% or above 20%.

Inhibition results

Percentage inhibition of growth rate (total test period) during the final test

 

Copaiba balsam oil Loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.760

0.0295

6

1

1.263

0.0733

3

28*

3.2

1.349

0.1099

3

23*

10

1.233

0.0091

3

30*

32

1.243

0.0306

3

29*

100

1.065

0.0209

3

40*

* Effect was statistically significant

Percentage inhibition of yield during the final test

Copaiba balsam oil loading rate

(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

196.0

17.83

6

1

44

10.05

3

78*

3.2

58.3

20.57

3

70*

10

39.4

1.11

3

80*

32

40.8

3.74

3

79*

.100

23.4

2.13

3

88*

* Effect was statistically significant

 

Validity criteria fulfilled:
yes
Remarks:
See details on results
Conclusions:
Under the conditions of this study with Pseudokirchneriella subcapitata, Copaiba balsam oil reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 1.0 mg/L and higher. However, a flat dose response curve was observed, which means that no EL10, EL20 and EL50 or NOEL could be calculated.

The EL50 for growth rate inhibition (72h-ERL50) was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L. The EL50 for yield inhibition (72h-EYL50) was lower than a loading rate of 1.0 mg/L. The 72h-NOEL for both growth rate inhibition and yield inhibition was lower than a loading rate of 1.0 mg/L.
Executive summary:

A full OECDTG 201 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 rates of 1.0, 3.2, 10, 32 and 100 mg Copaiba balsam oil per litre.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 with adjusted medium. Test vessels were air-tight closed vessels and headspace reduced to minimum. 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. Copaiba balsam oil reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 1.0 mg/L and higher. However, a flat dose response curve was observed. The EL50 for growth rate inhibition (72h-ERL50) was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L. The EL50 for yield inhibition (72h-EYL50) was lower than a loading rate of 1.0 mg/L. The 72h-NOEL for growth rate inhibition and yield inhibition was lower than a loading rate of 1.0 mg/L.

Description of key information

A full OECDTG 201 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 rates of 1.0, 3.2, 10, 32 and 100 mg Copaiba balsam oil per litre.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 with adjusted medium. Test vessels were air-tight closed vessels and headspace reduced to minimum. 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. Copaiba balsam oil reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 1.0 mg/L and higher. However, a flat dose response curve was observed. The EL50 for growth rate inhibition (72h-ERL50) was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L. The EL50 for yield inhibition (72h-EYL50) was lower than a loading rate of 1.0 mg/L. The 72h-NOEL for growth rate inhibition and yield inhibition was lower than a loading rate of 1.0 mg/L.

Key value for chemical safety assessment

Additional information