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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - October 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted under CLP conditions and in acc. with OECD guideline No. 203.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

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
- Test substance storage : in refrigerator in the dark
- Stability under storage conditions : stable
- Stability in water : unknown

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

other: Cyprinus Caproi, Teleostei, Cyprinidae

Details on test organisms:

-Source :
Zodiac, proefacc, "De Haar Vissen", L.U. Wageningen, the Netherlands.

- Mean length
Range-finding test: 2.78 ± 0.23 cm
Flow-through limit test: 3.43 ± 0.29 cm

- Mean weight
Range-finding test: 0.59 ± 0.14 g
Flow-through limit test: 1.08 ± 0.24 g

- Characteristics
F1 from a single parent-pair bred in UV-treated water.

- Reason for selection
This system has been selected as an internationally accepted species.

- Total fish used : 26

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

Daily in both vessels, beginning at the start of the test (day 0)

pH:

Daily in both vessels, beginning at the start of the test (day 0)

Dissolved oxygen:

Daily in both vessels, beginning at the start of the test (day 0)

Nominal and measured concentrations:

The sampling scheme during the flow-through study was as follows:

Frequency
2 1/2 hours before start of the exposure to check the test concentration, at the start of exposure, after 48 hours and at the end of the test period.

Volume Storage

10 ml from the approximate centre of the test vessels. Samples not analysed on the day of sampling were stored in a deep-freeze.

Additionally, reserve samples of 10 ml were taken. These samples were stored in a deep-freeze for possible analysis until delivery of the final report with a maximum of three months. The method of analysis is described in the appended Analytical Report.

Details on test conditions:

- Test duration : 96 hours

- Test type : Flow-through

- Test vessels
30 litres (50x30x25 cm) consisting of glass plates sealed with a thin film of silicone and covered by a removable Perspex plate.

- Test medium : Tap-water (data of analysis are attached).

- Number of fish : 7 fish per concentration and control.

- Loading : ca. 0.011 g fish/litre/day.

- Illumination : 16 hours photoperiod daily

- Aeration : The test media were not aerated during the test.

- Feeding : No feeding from 48 hours prior to the test and during the total test period.

- Introduction of fish
22 hours after the start of the dosing system to allow stabilisation of the test concentration.

-Mortality and other effects
At 3 1/2, 24, 48, 72 and 96 hours following the start of exposure. Dead fish were removed when observed.

- Fish length and weight
Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

- Euthanasia
At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Reference substance (positive control):

yes

Remarks:

Tap water without test substance

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Details on results:

- Static range-finding test :
No effect were observed in any of the concentrations tested. The LC50 was expected to be above 100 mg/l.
The analytical results showed that a shikimic acid concentration of 100 mg/l decreased by more than 20 % during the first 24 hours of exposure. As a consequence of the instability in the test medium further testing was performed applying continuous renewal of test solutions in a flow-through system.

- Final limit study (flow-through)

Measured concentrations

The analytical results showed an initial concentration of 111 mg/l. After 48 hours the concentration measured was 121 mg/l and after 96 hours the concentration measured was 104 mg/l. Based on these data the mean exposure concentration corresponded well with the target concentration of 100 mg/l (112 mg/l ± 5%).

Mortality and other effects

Exposure for 96 hours to an exposure concentration of 112 mg/l induced no mortality of fish.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

No mortality was observed in the control group. The analytical program confirmed that the actual concentration had been maintained at the target concentration ± 20%. All test conditions (pH, dissolved oxygen and temperature) remained within the ranges.

Conclusions:

Under the conditions of the present flow-through test SHIKIMIC ACID induced no visible effects in carp at an exposure concentration of 112 mg/l ± 5%.
The 96h-LC50 of SHIKIMIC ACID for fish exceeded the regulatory limit concentration of 100 mg/l.

Executive summary:

The test substance was completely soluble in test medium at the concentrations tested. The project started with a static range-finding test exposing carp to test concentrations of 0.1, 1.0, 10 and 100 mg/l. No visible effects were observed during the test in any of the concentrations tested. Analysis of samples taken during the range-finding test showed that a Shikimic Acid concentration of 100 mg/l decreased by more than 20% during the first 24 hours of exposure. As a consequence of the instability in the test medium further testing was performed applying continuous renewal of test solutions in a flow-through system. The experimental design was optimised by introducing continuous dosing of the test solution in a flow-through system. The target concentration for the final test was 100 mg/l. Exact volumes of a 20 mg/ml stock solution in Milli-RO water were dosed directly by a Gilson Dispenser via a computer­ controlled system. The dosed volume entered a mixing flask separately from the tap water supply. The tap water was supplied via a flow meter with a flow rate of ca. 12 l/h.

In the mixing flask, the dosed volume and the tap water were mixed under continuous stirring.

No mortality was observed in the control group.

Exposure to the Shikimic Acid concentration of 100 mg/l induced no visible effects or mortality of fish.

In conclusion, Shikimic Acid induced no visible effects in carp at an exposure concentration of 112 mg/l.

The 96h-LC50 of Shikimic Acid for fish exceeded the regulatory limit concentration of 100 mg/l.

The study procedures described in this report were based on the EEC directive 92/69, Part C.I. "Acute toxicity for fish"; and the OECD guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992.