6-aminopenicillanic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-11-03 to 2009-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted to current guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Variation in pH with the addition of the test material was not limited

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Variation in pH with the addition of the test material was not limited

GLP compliance:

yes (incl. QA statement)

Remarks:

In compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Controls: Untreated controls and a positive control (Harlan Laboratories Ltd Project No: 0039/1088) with potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source : Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/mL.

ACCLIMATION
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ± 1°C. The temperature within the incubator was recorded daily. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

Measured concentrations were used. Due to the rapid loss of the test material geometric means of the test samples were recorded.
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 - R3 pooled:
GM = √C0 x C1
where
GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type : closed
- Initial cells density: initial nominal cell density of 4 x 103 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Medium used: Please refer to table 2 for growth medium composition.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elga Optima 15+ or Elga Purelab Option R-15 BP

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study: A range finding study was performed over 72 hours
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: The results showed no significant effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/L. Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

55 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 54-57 mg/L 95 % CL

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

48 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

51 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

21 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 39-53 mg/L 95% CL

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 42-45 mg/L 95% CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/L, however few intact cells were observed to be present in the test cultures at 100 mg/L.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 6.25, 12.5 and 25 mg/L test cultures were observed to be pale green dispersions. The 50 mg/L test cultures were observed to be very pale green dispersions whilst the 100 mg/L test cultures were observed to be clear colourless solutions.
- Any stimulation of growth found in any treatment: None

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: Rate of growth EC50 (0 – 72 h):0.79 mg/L
Yield EC50 (0 – 72 h): 0.30 mg/L, 95% confidence limits 0.27 – 0.34 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

All tabulated data is included in the attached Pdf, Appendix I.

Validity criteria fulfilled:

yes

Remarks:

With the exception of the drift in pH the validation criteria set out in the methods was met.

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:
Growth Rate EC50 (mg/L): 55 (95% Confidence Limits (mg/L) 54-57)
Growth Rate No Observed Effect Concentration (NOEC) (mg/L) 25
Growth Rate Lowest Observed Effect Concentration (LOEC) (mg/L): 50
Yield EC50 (mg/L): 45 (95% Confidence Limits (mg/L) 39-53)
Yield No Observed Effect Concentration (NOEC) (mg/L) 25
Yield Lowest Observed Effect Concentration (LOEC) (mg/L): 50
The effect of the test material on the growth of Desmodesmus subspicatus based on the geometric mean measured test concentrations gave the following results:
Growth Rate EC50 (mg/L): 43 95% Confidence Limits (mg/L) 42-45
Growth Rate No Observed Effect Concentration (NOEC) (mg/L) 14
Growth Rate Lowest Observed Effect Concentration (LOEC) (mg/L): 38
Yield EC50 (mg/L): 32 (95% Confidence Limits (mg/L) 27-39)
Yield No Observed Effect Concentration (NOEC) (mg/L) 14
Yield Lowest Observed Effect Concentration (LOEC) (mg/L): 38

Description of key information

Toxicity to aquatic algae of 6-Aminopencillanic acid  was determined by OECD Guideline 201 (Alga, Growth Inhibition Test).

Additional information

The effect of 6-Aminopenicillanic acid on the growth of Desmodemus subspiculus is attributed to the physicochemical property of the substance and not its toxic property.

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:

Growth Rate EC50 (mg/L): 55 (95% Confidence Limits (mg/L) 54-57)

Growth Rate No Observed Effect Concentration (NOEC) (mg/L) 25

Growth Rate Lowest Observed Effect Concentration (LOEC) (mg/L): 50

Yield EC50 (mg/L): 45 (95% Confidence Limits (mg/L) 39-53)

Yield No Observed Effect Concentration (NOEC) (mg/L) 25

Yield Lowest Observed Effect Concentration (LOEC) (mg/L): 50

The effect of the test material on the growth of Desmodesmus subspicatus based on the geometric mean measured test concentrations gave the following results:

Growth Rate EC50 (mg/L): 43 95% Confidence Limits (mg/L) 42-45

Growth Rate No Observed Effect Concentration (NOEC) (mg/L) 14

Growth Rate Lowest Observed Effect Concentration (LOEC) (mg/L): 38

Yield EC50 (mg/L): 32 (95% Confidence Limits (mg/L) 27-39)

Yield No Observed Effect Concentration (NOEC) (mg/L) 14

Yield Lowest Observed Effect Concentration (LOEC) (mg/L): 38