D-gluconic acid, compound with N,N''-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine (2:1)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

cited as 92/69/EEC (C.2.) (1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Infracor Chemistry Services

Analytical monitoring:

yes

Details on sampling:

At the time of test performance no analytical method with adequate sensitivity was available to analyse the test substance concentration in the concentration range used for testing was available. Due to these facts, only the test substance concentrations of the stock solution and of an additional stability control (= test substance concentration directly above the detection limit of 1 mg TOC/L) could be determined after 0 and 48 hours by TOC-analysis.
To avoid negative effects due to the inorganic C-compounds, test batches used for the analytical determinations were prepared without addition of NaHCO3.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain: clone 5
- Source: Animals bred at the testing facility, originally derived from Dr. N. Caspers, Bayer AG, Leverkusen, Germany
- Age at study initiation: Neonates < 24 h old
- Method of breeding: For breeding the females were maintained in M4-medium in 1L beakers.
At two to three day intervals, the exuviae were syphoned off, the water was changed and the offspring removed from the breeding vessels.
At regular intervals of approximately 4 weeks, offspring was isolated from the vessels. They served as a basis for further breeding.
Females are separated from the offspring 24 hours before the start of the test. The litter produced overnight was used for the test.
The culture conditions ensure parthenogenetic reproduction.
- Feeding during test: no

ACCLIMATION
- Food type: Desmodesmus (formerly Scenedesmus) subspicatus, unicellular green algae
- Amount: Amount of food was such that the amount added was consumed by the animals
- Frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

14°dH = 250 mg CaCO3/L

Test temperature:

20 +/- 1°C

pH:

7.7 - 8.0

Dissolved oxygen:

7.6 - 8.3 mg/L

Nominal and measured concentrations:

0.0077, 0.013, 0.023, 0.040, 0.070, 0.12, 0.21 mg/L (concentration are based on the main product)
The measured recovery rate of the stock solution concentrations and stability control concentration after 48 hours did not deviate by more than 20 % from the freshly prepared solutions.
It can be concluded that the test substance concentration was stable during the whole exposure time. Therefore the nominal concentration of the stock solution was used to calculate the test substance concentrations in the test batches.

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 mL
- Type: open. Test substance is not volatile
- No. of organisms per vessel: 5 +/- 1
- No. of vessels per concentration (replicates): 4
- Biomass loading rate: Approx. 2 mL per test organisms

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic fresh water
- Ca/Mg ratio: 4 : 1
- Culture medium different from test medium: Yes, Elendt M4 medium as described in OECD Guideline 202

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Complete darkness

EFFECT PARAMETERS MEASURED : Immobility. Sampling at 24 and 48 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.087 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 0.07-0.12 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? yes
EC50 (24 h) > 1 mg/L (nominal concentrations)
EC100 (24 h) = 2 mg/L

Reported statistics and error estimates:

EC50 -values were determined graphically

 

	EC50	95 % c.l.	EC0	EC100
24h [mg/L]	> 0.21	no data	0.07	> 0.21
48h [mg/L]	0.087	0.07-0.12	0.04	0.12

(concenrations are based on a.i.)

Although no substance specific analytical measurement was performed in the OECD202 study, the effect value retrieved based on nominal concentrations is plausible and acceptable. This is supported by the OECD211 study which contains substance specific analytical monitoring.

The OECD211 study was performed as a semi-static test design with 3 times per week test medium renewal. Adult mortality occurred only in the highest test concentration (0.3 mg test item/l corresponding to 61.8 µg a.i. CHDG/l) and started after 5 days of incubation (10% mortality). Analytical monitoring indicated that the substance concentration was sufficiently maintained.

Based on the analytical results and adult mortality results from the OECD211 study it can be concluded for an OECD202 study that the substance concentration should be maintained within a period of 48h and a 48h EC50 acute toxicity value should clearly be >61.8 µg a.i. CHDG/l. This indicate that the available OECD202 study is reliable and the available nominal effect value of 87 µg a.i. CHDG/l is plausible and acceptable as an acute effect value.

Validity criteria fulfilled:

yes

Conclusions:

The test results indicate that Chlorhexidine digluconate is acutely very toxic towards daphnia. The 48h-EC50 was determined to be 0.087 mg a.i./L.

Description of key information

Acute toxicity towards invertebrates was tested with the water flea Daphnia magna, and the EC50 was established at 0.087 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.087 mg/L