6-aminopenicillanic acid

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:

2009-11-2 to 2009-11-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted to current accepted guidelines.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/L test group (replicates R1 – R2 and R3 – R4 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the required test concentration of 100 mg/L.
- Controls: An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/L stock solution. An aliquot (50 mL) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/L stock solution. Aliquots (16, 28, 50, 90 and 160 mL) of the 10 mg/L stock solution were each separately dispersed in a final volume of 500 mL of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna
- Source: in-house laboratory cultures.
- Age at study initiation : 1st instar daphnids, less than 24 hours old
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: The daphnids received no food during exposure. Prior to the test, each culture was fed daily with a suspension of algae (Chlorella sp.).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Physico-Chemical measurements of the test media are tabulated in table 1.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Dissolved oxygen:

Physico-Chemical measurements of the test media are tabulated in table 1.

Nominal and measured concentrations:

Analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 89% to 109% of nominal value and so the results are based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Type : Closed, covered to reduce evaporation
- Material, size, fill volume: 250 mL polypropylene vessels
- Aeration: the test vessels were not aerated.
- Type of flow-through: The preliminary stability analyses conducted for the Algal Growth Inhibition Test (Harlan Laboratories Ltd Project Number 1127/1850) indicated that the test material was unstable. Therefore, as a precautionary measure semi-static test conditions were employed in this test. For the test media renewal at 24 hours, the 100 mg/l test concentration was freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 25 mL aliquots of the following were added to deionised water; CaCl2.2H2O 11.76 g/L, MgSO4.7H20 4.93 g/L, NaHCO3 2.59 g/L and KCl 0.23 g/L; with a final volume of one litre.
- Conductivity: < 5 µS cm-1
- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.
- Intervals of water quality measurement: 0, 24 and 48 hours

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl during preparation. While there were no treatment related differences for oxygen concentration the pH of the 100 mg/L test vessels was observed to be significantly different from that of the control vessels. This was considered to be due to an intrinsic property of the test material and as such no alteration was made to the pH prior to exposure.
- Photoperiod: 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Range finding study: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations. 10 daphnids were tested in each control and test vessel.
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg/L in the range finding study, in the main test 0 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/L. A concentration of 100 mg/L was employed in the main test.

POSITIVE CONTROL:
A positive control (Harlan Laboratories Ltd Project No: 0039/1094) conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Harlan Laboratories Ltd Project No: 0039/1094) conducted approximately every six months

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

Immobilisation

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

Immobilisation

Details on results:

- Mortality of control: At 48 hours, one immobilisation (5%) was noted in the control.
- Abnormal responses: No abnormal responses were noted in the definitive test. No daphnids were immobilised in the presence of the test material either in the range-finder or the definitive test.

All tabulated results are presented in the attached pdf, Appendix I.

Results with reference substance (positive control):

- Results with reference substance valid?
- Mortality: At 0.56 mg/L the mortality was 5 %, 1.0 mg/L 90%, and at 1.8 mg/L and above the mortality was 100 %. After 24 hours in the highest dosing group, the mortality was recorded as 100%. The NOEC was 0.32 mg/L.
- EC50/LC50: The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.20).

Reported statistics and error estimates:

For the positive control, the EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curves and standard errors were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.

Table 2: Physico-chemical measurement of test media

 

Nominal concentration (mg/L)		0 hours (fresh media)				24 hours (old media)				24 hours (fresh media)				48 hours (old media)
		pH	mg O2/L	%ASV*	T°C	pH	mg O2/L	%ASV*	T°C	pH	mg O2/L	%ASV*	T°C	pH	mg O2/L	%ASV*	T°C
Control	R1	8.0	9.1	100	20	8.0	8.8	101	21	7.7	9.0	99	20	7.6	8.9	102	22
	R2	8.0	9.1	100	20	8.0	8.9	102	21	7.7	9.1	100	20	7.6	8.7	100	22
	R3	8.0	9.0	99	20	7.8	9.0	103	21	7.7	9.1	100	20	7.6	8.6	97	21
	R4	8.0	9.1	100	20	7.9	9.1	105	21	7.7	9.2	101	20	7.6	8.6	97	21
100	R1	6.2	9.1	100	20	6.9	9.3	107	21	5.6	9.2	101	21	5.8	8.7	100	22
	R2	6.1	9.1	100	20	6.3	9.4	108	21	5.6	9.3	102	21	5.7	8.7	100	22
	R3	6.1	9.1	100	20	6.3	9.4	108	21	5.6	9.3	102	21	5.7	8.7	98	21
	R4	6.1	9.1	100	20	6.3	9.4	108	21	5.6	9.2	101	21	5.7	8.8	99	21
*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value R1-R4= Replicates 1 to 4

Validity criteria fulfilled:

yes

Remarks:

The study fulfils all of the criteria set out in the OECD guideline.

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/L. Correspondingly the No Observed Effect Concentration was 100 mg/L.

Description of key information

Short term toxicity to aquatic invertebrates of 6-Aminopenicillanic acid was determined by OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test).

Additional information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/L. Correspondingly the No Observed Effect Concentration was 100 mg/L.