(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

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:

May - August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted under CLP conditions and in acc. with ISO International Standard 8692.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

EC Number:

205-334-2

EC Name:

(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

Cas Number:

138-59-0

Molecular formula:

C7H10O5

IUPAC Name:

(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

Details on test material:

- Description : white powder
- Purity : 98,9 %
- Test substance storage : in refrigerator in the dark
- Stability under storage conditions : stable
- Expiry date : 3 March 2002

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test samples were taken from three concentrations, i.e. 2.2, 22 and 100 mg/l and
the blank-control.

Frequency : at t=O h, t=24 h and t=72 h
Volume : 10 ml
Storage : Samples were transferred to Analytical Chemistry directly after sampling.

Test solutions

Vehicle:

yes

Details on test solutions:

M2-medium according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using
Milli-Q water preventing precipitation and with the following composition:

NH4CI 15 mg/l
MgCI2.6H20 12 mg/l
CaCI2.2H20 18 mg/l
MgS04.7H20 15 mg/l
KH2P04 1.6 mg/l
FeCI3.6H20 80 µg/l
Na2EDTA.2H20 100 µg/l
H3B03 185 µg/l
MnCI2.4H20 415 µg/l
ZnCI2 3 µg/l
CoCI2.6H20 1.5 µg/l
CuCI2.2H20 0.01 µg/l
Na2Mo04.2H20 7 µg/l
NaHC03 50 mg/1
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Species : Selenastrum capricornutum, strain: CCAP 278/4.

- Reason for selection : This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Every day in a temperature-control vessel.

pH:

At the begining and at the end if the test.
The pH of the solution should preferably not deviate by more than 1,5 units during the test.

Details on test conditions:

- 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 (4000-9000 lux) in a climate room at a temperature of 23 ± 2°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 2x 10000 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

- Concentrations : 2.2, 4.6, 10, 22, 46 and 100 mg/l

- Controls : Test medium without test substance or other additives (blank).

- Replicates
3 replicates of each test concentration.
6 replicates of the blank-control.
1 replicate of each concentration without algae. extra replicates of each test concentration for sampling purposes.

Reference substance (positive control):

yes

Remarks:

Test medium without test substance or other additives.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 48 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Details on results:

1. In the controls, cell density increased by an average factor of > 16 within three days.

2. Analysis of samples taken during the study showed that the actual exposure concentrations could not be maintained above 80% relative to theinitial concentration. This could not be prevented, as the experimental design does not allow for intermittent renewal. However, the current study included exposure to the degradation products, which will also occur in the aquatic environment after a possible discharge or spill.

3. Further, all test conditions remained within the ranges prescribed by the protocol, except for the pH in some of the treated groups. The pH increased by more than 1.5 unit, but this was related to a relative high rate of algal growth. Hence, this deviation did not affect the validity criterion for algal growth.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, SHIKIMIC ACID reduced growth rate of this fresh water algae species significantly at nominally 100 mg/l, corresponding with an average measured exposure concentration of 34 mg/l.

The EC50 for both growth rate reduction (ERC50: 0-72h) and cell growth inhibition (E8C50: 0-72h) were beyond the range tested, i.e. above nomnally 100 mg/l corresponding with an average exposure concentration of 34 mg/l.

The NOEC for both cell growth inhibition and growth rate reduction were at nominally 46 mg/l, corresponding with an average exposure concentration of 16 mg/l.

Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with SHIKIMIC ACID.

The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989.

The batch of SHIKIMIC ACID tested was a white powder with a purity of 98.9% and the substance was completely soluble in test medium at the concentrations tested.

After a range-finding test, a final EC₅₀ test was performed exposing exponentially growing algal cultures to nominal SHIKIMIC ACID concentrations of 2.2, 4.6, 10, 22, 46 and 100 mg/l. The study further included a blank-control. The initial cell density was 10⁴ cells/ml. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours and at the end of the 72-hour test period.

Analysis of the samples taken at the start of the final test showed that the actual test concentrations were in agreement with nominal (91-102%). After 24 hours of exposure measured concentrations at 4.6 to 100 mg/l had remained stable and in agreement with nominal (90-102%). However, the measured concentration at nominally 2.2 mg/l had decreased to 0.6 mg/l, i.e. by approximately 70% below initial. At the end of the 72-hour test period no detectable test substance concentrations were found in the samples taken from 2.2, 22, 46 and 100 mg/l (<0.04 mg/l). Measured concentrations at nominally 100 mg/l without algae had decreased to 0.13 mg/l. The average exposure concentrations at the concentrations essential for determination of the effect parameters, i.e. 46 and 100 mg/l, were 16 and 34 mg/l, respectively.

SHIKIMIC ACID reduced growth rate of this fresh water algae species significantly at nominally 100 mg/l, corresponding with an average measured exposure concentration of 34 mg/l.

The EC₅₀ for both growth rate reduction (E_RC₅₀: 0-72h) and cell growth inhibition (E_BC₅₀: 0-72h)

were beyond the range tested, i.e. above nominally 100 mg/1 corresponding with an average exposure

concentration of 34 mg/1.

The NOEC for both cell growth inhibition and growth rate reduction were at nominally 46 mg/l,

corresponding with an average exposure concentration of 16 mg/l.