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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
deviation is considered not to affect the integrity of the study or the validity of the conclusions drawn.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The test substance was received at Brixham Environmental Laboratory on 19 May 2004 and assigned the Brixham test substance number 04-0169. The test substance (Batch No SP04027/GLP) was supplied as a white to cream solid. The sample was stored at ambient temperature, in the container in which it was received until required for testing, when an appropriate subsample was provided for the test operator.
Analytical monitoring:
yes
Vehicle:
not specified
Details on test solutions:
This study was run with a dilution water control together with a nominal concentration of ceftazidime dihydrochloride of 120 mg L-1. A 1000 ml volume of the nominal 120 mg r1 test solution was prepared by the direct addition of ceftazidime dihydrochloride to dilution water. This solution was stirred for approximately 5 minutes. The resultant solution was clear and colourless. The control consisted of dilution water only.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test species was the freshwater crustacean, Daphnia magna, obtained from continuous laboratory cultures. The stock cultures of Daphnia were maintained in a reconstituted water medium, identical to the test dilution water, at a temperature of 20 ± 1 °C and in 2 litre glass vessels with a working volume of 1.5 litres. A photoperiod of 16 hours light:8 hours dark, with 20 minute dawn and dusk transition periods was provided. The Daphnia cultures were fed a defined diet of algae Chlorella vulgaris, strain CCAP 211/12 and a commercially available fish food. Culture conditions were such that the Daphnia reproduction was by diploid parthenogenesis . Daphnia <24 hours old (first instar), obtained from a single culture vessel, were used for testing. The parent animals were 27 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium. The test organisms and the culture from which they were obtained showed no evidence of disease before the test period .
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The total hardness of the Daphnia media was 239 mg L-1 CaC03
Test temperature:
20 ± 1°C
pH:
6.79 to 8.18
Dissolved oxygen:
8.2 to 9.4 mg L-1
Conductivity:
the conductivity of the water was 654 µS cm-1
Nominal and measured concentrations:
nominal concentration of 120 mg L-1

The overall mean measured concentration of ceftazidime dihydrochloride was 100% of the nominal exposure concentration .
Details on test conditions:
Test procedure and apparatus
Borosilicate glass beakers of 250 ml nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 ml of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The positions of the treatments were randomly allocated within the test area. The test was initiated by the addition of five randomly selected Daphnia, in <2.0 ml of dilution water, to each test vessel in sequence across the treatments within 10 minutes of the test solution preparation. Each treatment contained a total of 20 Daphnia. The loading of the Daphnia in each test vessel was 25 Daphnia L-1. The nominal test solution temperature was 20 ± 1 °C, maintained by control of the room temperature. A photoperiod of 16 hours light:8 hours dark, with 20 minute dusk and dawn transition periods, was provided. The test solutions were not aerated and the Daphnia were not fed during the course of the study.

Preparation of test solutions
This study was run with a dilution water control together with a nominal concentration of ceftazidime dihydrochloride of 120 mg L-1. A 1000 ml volume of the nominal 120 mg L-1 test solution was prepared by the direct addition of ceftazidime dihydrochloride to dilution water. This solution was stirred for approximately 5 minutes. The resultant solution was clear and colourless. The control consisted of dilution water only.

Analytical method
The concentration of ceftazidime dihydrochloride in the test solution was measured at 0 and 48 hours using the high performance liquid chromatography.

Observation of effects
An assessment of the response of the Daphnia was made 24 and 48 hours after the commencement of the test. Each Daphnia was viewed by eye and was defined as affected if showing no whole body movement, relative to the water, within a period of 15 seconds even if movement of individual appendages was visible. Daphnia that were affected were termed immobile. Any overt symptoms of toxicity were also recorded. The median effect concentration (EC50) is defined as the concentration resulting in 50% immobilisation of the Daphnia in the time period specified. The numbers of Daphnia immobilised in the replicates of the dilution water control and test concentrations were summed for each time period.

Physical and chemical parameters
The total hardness of the reconstituted dilution water was measured (as mg L-1 CaC03) before the start of the test.

Measurements were undertaken at the beginning and end of the test for pH and dissolved oxygen concentration using calibrated meters. The dissolved oxygen concentration of the dilution water control (dilution water used to prepare the test solutions) was measured using the excess medium remaining after filling the control vessels. The initial pH of the test solution was measured using the excess remaining after filling the test vessels. At the end of the study, the pH and dissolved oxygen concentration of two replicates of the dilution water control and the ceftazidime dihydrochloride test solution were measured.

Temperature values were determined daily using a mercury-in-glass thermometer calibrated to 0.1 °C and conforming to BS593. Hourly measurements were also recorded automatically in the additional dilution water control test vessel using a calibrated electronic recording system.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Analytical data
The concentration of ceftazidime dihydrochloride determined in the test solution is recorded in Table 1 in the attachment in the background section. The highest limit of quantification of ceftazidime dihydrochloride in this study was 0.047 mg L-1. All analytical values are quoted to two significant figures and percentages to the nearest integer. The overall mean measured concentration of ceftazidime dihydrochloride was 100% of the nominal exposure concentration . On the basis of the analytical data the nominal concentration of ceftazidime dihydrochloride was used for the reporting of results.

Biological data
No symptoms of toxicity or immobility were observed in this study.

The results obtained were:
Time EC50
24 hour > 120 mg L-1
48 hour > 120 mg L-1

The no observed effect concentration (NOEC) is defined as the highest tested concentration in which there was no immobility of the Daphnia within the period of the test, therefore,

48 hour NOEC = 120 mg L-1
The EC100 is defined as the lowest test concentration that caused 100% immobility within the period of the test, therefore,
48 hour EC100 = >120 mg L-1
No symptoms of toxicity or immobility were observed in the dilution water control.

Physical and chemical data
Dissolved oxygen concentrations ranged from 8.2 to 9.4 mg rt and the pH values ranged from 6.79 to 8.18. At no time during the course of the study was dissolved oxygen concentration in any of the test vessels less than 60% of the air-saturation value.

The thermometer readings at 0, 24 and 48 hours were 20.2, 20.4 and 20.2°C. The continuous temperature recorded automatically over the 48 hours remained within 20± 1°C.

The total hardness of the Daphnia media was 239 mg L-1 CaC03 and the conductivity of the water was 654 µS cm".
Validity criteria fulfilled:
yes
Conclusions:
No symptoms of toxicity or immobility were observed in this study.

The no observed effect concentration (NOEC) is defined as the highest tested concentration in which there was no immobility of the Daphnia within the period of the test, therefore,
48 hour NOEC = 120 mg L-1

The EC100 is defined as the lowest test concentration that caused 100% immobility within the period of the test, therefore,
48 hour EC100 = >120 mg L-1
Executive summary:

Test Species: Daphnia magna

Source of organisms: Continuous laboratory cultures; parental stock 27 ± 1 days old

Test concentrations: Dilution water control and nominal concentration of 120 mg l-1

Length of test: 48 hours, static

Nominal test temperature: 20 ± l°C

Results based on                     Time                     EC50

nominal concentration               24 hour                  >120 mg l-1

of Ceftazidime                          48 hour                  >120 mg l-1

dihydrochloride

48 hour no observed effect concentration (NOEC) based on immobility = 120 mg l-1

Description of key information

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
120 mg/L
EC50/LC50 for marine water invertebrates:
120 mg/L

Additional information

The LC50 was >120mg/L which was the highest nominal value tested