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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 to 19 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Deviation did not 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 Brixharn 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
Details on sampling:
After 24, 48, and 72 hours, samples were removed from each test and blank vessel to determine the algal absorbance

The concentrations of ceftazidime dihydrochloride in the test solutions were measured at 0 and 72 hours using the high performance liquid chromatography.
Samples for analysis were ta.ken at O hours from the excess test solutions and at 72 hours from the remaining blank solutions.
Vehicle:
yes
Remarks:
sterile culture medium
Details on test solutions:
This study was run with a dilution water control together with nominal concentrations of ceftazidime dihydrochloride of 0.0016, 0.0031, 0.0063, 0.013, 0.025, 0.050, 0.10 and 0.20 mg L-1. A 1000 ml volume of stock concentrate (40 mg r1) was prepared by direct addition of ceftazidime dihydrochloride to sterile culture medium. A 2000 ml volume of the highest nominal test concentration (0.20 mg L-1) was prepared from the stock concentrate added to sterile culture medium. This solution was stirred for approximately 15 minutes resulting in a clear, colourless solution. The lower test concentrations were prepared, using sterile culture medium, by addition of aliquots of the nominal 0.20 mg L-1 test solution to final volumes of 1000 ml. The solutions were stirred for approximately 15 minutes and were clear and colourless. The control consisted of culture medium only. In all cases the final solutions contained nutrients. Using aseptic techniques, 100 ml volumes of the appropriate test solution were dispensed to each test and blank vessel, with the remainder of the test solutions being used for physical and chemical analyses.
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
The test species was the blue green alga Anabaena flos-aquae (CCAP 1403/BA) from laboratory cultures maintained under axenic conditions. A 4 day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.
Test type:
not specified
Water media type:
other: Media was prepared according to Miller W E, Greene JC and Shiroyama T (1978). Selenastrum capricornutum Printz. Algal Assay Bottle Test: Experimental Design, Application and Data Interpretation Protocol. EPA-600/9-78-018, Corvallis, OR.
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 2°C
pH:
At the start of the test the pH ranged from 7.45 to 7 .60 and at the end of the test the range was from 7.39 to 8.05
Nominal and measured concentrations:
Culture medium control and nominal concentrations of 0.0016, 0.0031, 0.0063, 0.013, 0.025, 0.050, 0.10 and 0.20 mg L-1
The mean measured concentrations ranged from 84% to 95% of the nominal values.
Details on test conditions:
The test vessels were borosilicate glass conical flasks of 250 ml nominal capacity capped with polyurethane foam bungs. Each flask contained 100 ml of test solution. The cultures were incubated at 24 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination, with orbital shaking at 160 rpm, in a Gallenkamp type NR-401 orbital incubator . Six replicate cultures of the culture medium control and triplicate cultures of each concentration of ceftazidime dihydrochloride were employed. The positions of the test vessels in the incubator were randomised by rows, and re-randomised daily. One blank vessel (without algal inoculum) for the culture medium control and each test concentration was incubated concurrently . The cell density of the culture used as the inoculum for the test was determined by microscope counting using a haemocytometer chamber and was 3.96 x 106 cells mL-1. Each replicate test vessel was inoculated with 0.255 ml of the inoculum culture to give a nominal cell density of 1.00 x 10 4 cells mL-1 . The relative algal cell densities of the inoculum and test cultures were determined by spectrophotometric absorbance, using the method outlined in Appendix 2. If necessary, test samples were diluted to give measured absorbance values within the linear calibration range. At the start of the test, the absorbance of a range of dilutions of the inoculum culture was used to determine the relationship between absorbance and cell density. All results are quoted by absorbance. At the start of the study the algal absorbance was below the limit of detection of the method, consequently, the initial absorbance was calculated as 0.003 absorbance units . After 24, 48, and 72 hours, samples were removed from each test and blank vessel to determine the algal absorbance.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.013 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.025 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EbC50
Remarks:
Median effective concentration, biomass
Effect conc.:
ca. 0.024 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: 95 % confidence limit
Effect conc.:
ca. 0.018 - ca. 0.031 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Remarks:
Median effective concentration, biomass
Effect conc.:
ca. 0.053 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Based on logarithmic growth rate over the test period
Key result
Duration:
72 h
Dose descriptor:
other: 95 % confidence limit
Remarks:
Based on logarithmic growth rate over the test period
Effect conc.:
ca. 0.042 - ca. 0.065 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Analytical data
The concentrations of ceftazidime dihydrochloride determined in the test solutions are given in Table 1- attached in background section. The highest limit of quantification of ceftazidime dihydrochloride in this study was 0.00054 mg L-1. All analytical values are quoted to two significant figures and percentages to the nearest integer. The mean measured concentrations ranged from 84% to 95% of the nominal values. On the basis of the analytical data the nominal concentrations of ceftazidime dihydrochloride were used for the calculation and reporting of the results.

Biological data
The spectrophotometric absorbances measured at each time period are given in Table 2 - attached in background section. The means of these values are also shown in Table 2- attached in background section and are plotted as growth curves in Figure 1- attached in background section.

Areas under the growth curve
The area under the growth curve (0 to 72 hours) was calculated for each replicate culture, according to the formula given in the OECD Guideline. These areas were examined by one-way analysis of variance, and Dunnett's procedure was used to identify significant differences (P=0.05) from the culture medium control. The mean areas under the growth curve, and the significant differences identified, are given in Table 3 - attached in background section, together with the areas expressed as percentages of the culture medium control.

A Weibull model was fitted to the percent effect and log concentration data; this model was then used to estimate the median effective concentration (based on biomass curve area, EbC50) and its 95% confidence limits. The results obtained from these statistical analyses, based on mean measured test concentrations, were as follows:
No observed effect (P==0.05) concentration (NOEC) = 0.013 mg L-1
Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mg L-1
Median effective concentration, biomass (EbC50) = 0.0242 mg L-1
95% confidence limit = 0.0177 - 0.0307 mg L-1

Growth rates
The growth rate (0 to 72 hours) was calculated for each replicate culture, according to the formula.
Growth rate = [(Logn(N2/N1)]/t
Where N1 = Relative absorbance at start of test
N2 = Relative absorbance at end of test
t = Time interval (days)

These data were analysed as described for the area method. The mean growth rates, and the significant differences identified, are given in Table 4, together with the rates expressed as percentages of the culture medium control. The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:
No observed effect (P=0.05) concentration (NOEC) = 0.013 mg L-1
Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mg L-1
Median effective concentration, biomass (ErC5O) = 0.0535 mg L-1
95% confidence limit = 0.0419 - 0.0651 mg L-1

Physical and chemical data
At the start of the test the pH ranged from 7.45 to 7 .60 and at the end of the test the range was from 7.39 to 8.05 (Table 5) - attached in background section.

The daily temperature measurements recorded by thermometer in the incubator ranged from 23.9 to 24.0°C. The hourly temperature measurements, recorded automatically, remained within 24 ± 2°C. The light intensity, measured once during the study, was 7190 lux (by cosine receptor). This was also measured in terms of quantum response and was 90.5 µ, Einsteins m-2 s-1.
Reported statistics and error estimates:
A Weibull model was fitted to the percent effect and log concentration data; this model was then used to estimate the median effective concentration (based on biomass curve area, EbC50) and its 95% confidence limits.
Validity criteria fulfilled:
yes
Conclusions:
Results based on nomimal concentrations of Ceftazidime dihydrochloride: Based on areas under the growth curve the results obtained at 72 hours were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1
Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1
Median effective concentration, biomass (EbC50) = 0.0242 mgL-1
95% confidence limit = 0.0177 - 0.0307 mgL-1

Based on logarithmic growth rate over the test period, the results were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1
Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1
Median effective concentration, biomass (ErC50) = 0.0535 mgL-1
95% confidence limit = 0.0419- 0.0651 mgL-1
Executive summary:

Source of test organisms: Brixham Environmental Laboratory culture from strain CCAP 1403/L3A

Test concentrations: Culture medium control and nominal concentrations of 0.0016, 0.0031, 0.0063, 0.013, 0.025, 0.050, 0.10 and 0.20 mg L-1

Length of test: 72 hours shaken

Test dates: 16 to 19 August 2004

Nominal test temperature: 24 ± 2°C

Results based on nomimal concentrations of Ceftazidime dihydrochloride: Based on areas under the growth curve the results obtained at 72 hours were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1

Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1

Median effective concentration, biomass (EbC50) = 0.0242 mgL-1

95% confidence limit = 0.0177 - 0.0307 mgL-1

Based on logarithmic growth rate over the test period, the results were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1

Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1

Median effective concentration, biomass (ErC50) = 0.0535 mgL-1

95% confidence limit = 0.0419- 0.0651 mgL-1

Description of key information

Results based on nomimal concentrations of Ceftazidime dihydrochloride: Based on areas under the growth curve the results obtained at 72 hours were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1

Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1

Median effective concentration, biomass (EbC50) = 0.0242 mgL-1

95% confidence limit = 0.0177 - 0.0307 mgL-1

Based on logarithmic growth rate over the test period, the results were:

No observed effect (P=0.05) concentration (NOEC) = 0.013 mgL-1

Lowest observed effect (P=0.05) concentration (LOEC) = 0.025 mgL-1

Median effective concentration, biomass (ErC50) = 0.0535 mgL-1

95% confidence limit = 0.0419- 0.0651 mgL-1

Key value for chemical safety assessment

EC50 for freshwater algae:
0.024 mg/L
EC50 for marine water algae:
0.024 mg/L
EC10 or NOEC for freshwater algae:
0.013 mg/L
EC10 or NOEC for marine water algae:
0.013 mg/L

Additional information