Cypress, Cupressus funebris, ext.

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Cypress, Cupressus funebris, ext.

EC Number:

285-360-9

EC Name:

Cypress, Cupressus funebris, ext.

Cas Number:

85085-29-6

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood obtained from the wood of Cupressus funebris (Cupressaceae) by steam distillation

Test material form:

other: Pale yellow to yellow liquid

Remarks:

could crystallize

Details on test material:

- Name of test material (as cited in study report): Cedarwood Oil China
- 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: Cedarwood Oil China
Appearance: Pale yellow to yellow liquid
Batch: 1002515317
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2017 (expiry date)

Test facility test item number: 207805/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Chamaecyparis funebris (Cupressaceae) obtained from the wood by steam distillation
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility water: Not available
Stability in water: Not available

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.
Analysis set-up No. of repeats: at least 3

Test solutions

Vehicle:

no

Remarks:

Water Accomodated Fractions (WAFs) were used

Details on test solutions:

The batch of Cedarwood Oil China tested was a pale yellow to 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 0.16 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 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.
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 40 mL 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.
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.

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 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°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: 0.16, 0.8, 4.0, 20, 100 mg/L
TOC measurements:
TOC t=0h : n.q., 0.63, 0.29, 2.43, 7.60 mg TOC/L
TOC t=72h: n.q., 0.17, 0.18, 2.63, n.d. mg TOC/L

The TOC content of the test item was estimated to be 82.7%

n.q.: not quantifiable
n.d.: not determined

Details on test conditions:

Cedarwood Oil China: WAFs prepared at loading rates of 0.16, 0.80, 4.0, 20, 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 buffer 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 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 85 to 86 µE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

A spacing factor higher than 3.2 was used as the dose-response observed in the range-finding test was very shallow.

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 0.46, 1.0, 10 and to 100 mg/L.
• Three replicates were tested per concentration and three replicates in the control group.
• 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 examined for their appearance.

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 background for the treated solutions.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

116 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 101 - 138

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 7.2 - 12

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on biological significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 7.1 - 9.3

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.17 - 0.31

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

An increase in the measured concentration was observed at the start of the test indicating proper preparation of the WAFs. It should be noted, that values below 1.0 mg TOC/L should be considered indicative. The concentration measured in WAF of 100 mg/L was 7.6 mg/L and was comparable to the result in the range-finding test, showing repeatability of the preparation procedure.
Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of initially prepared loading rates.

Inhibition of growth rate increased from 1.0% at the lowest to 48% in the highest WAF tested. The inhibition recorded at the highest WAF was similar to the observed in the range-finding test in the same treatment. A statistically significant effect was observed in the WAFs of 0.80 mg/L and higher, therefore, the NOEL was 0.16 mg/L. Because the effects observed at 0.80 and 4.0 mg/L were considered biologically insignificant (<10%), the NOEL based on biological significance was set to 4.0 mg/L.
Inhibition of yield increased with applied dose, from 5.8% at the lowest to 92% in the highest WAF tested. A statistically significant effect was observed in the WAFs of 0.80 mg/L and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to WAF of 20 mg/L when compared to the control.

Validity criteria:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 228).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 14%).
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):

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 (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) 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 max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rate 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

CW OIL CHINA Loading rate (mg/L)	%Inhibition
Control
0.16	1.0^#
0.80	8.4^#
4.0	5.3^#
20	16*
100	48*

* - effect was statistically significant,^#- effect was statistically significant but not biologically relevant

Yield (x10⁴Cells/mL) And Percentage Inhibition For The Total Test Period

CW OIL CHINA Loading rate (mg/L)	%Inhibition
Control
0.16	5.8
0.80	37*
4.0	26*
20	58*
100	93*

* - effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See details on results

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Cedarwood Oil Chinese reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 0.80 mg/L and higher.
The EL50 for growth rate inhibition (72h-ErC50) exceeded the range tested and was estimated using regression method to be 116 mg/L with a 95% confidence interval ranging from 101 to 138 mg/L.
The EL50 for yield inhibition (72h-EyC50) was 8.1 mg/L with a 95% confidence interval ranging from 7.1 to 9.3 mg/L.
The 72h-NOEL for growth rate inhibition was 4.0 mg/L based on biological relevance and 0.16 mg/L based on statistical significance.
The 72h-NOEL for yield inhibition was 0.16 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 rates of 0.16, 0.80, 4.0, 20 and 100 mg Cedarwood Oil Chinese 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. 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. An increase in the measured TOC concentration was observed at the start of the test indicating proper preparation of the WAFs. In conclusion, Cedarwood Oil Chinese reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 0.80 mg/L and higher. The EL50 for growth rate inhibition (72h-ErL50) exceeded the range tested and was estimated using regression method to be 116 mg/L with a 95% confidence interval ranging from 101 to 138 mg/L. An ErL10 was concluded at 9.7 mg/L (7.2 - 12 mg/L; 95% CI). The 72h-NOEL for growth rate inhibition was 4.0 mg/L based on biological relevance and 0.16 mg/L based on statistical significance.

The EL50 for yield inhibition (72h-EyL50) was 8.1 mg/L with a 95% confidence interval ranging from 7.1 to 9.3 mg/L. The 72h-NOEL for yield inhibition was 0.16 mg/L.