Pyridinium, 1-[[(6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-, chloride, hydrochloride (1:1:1)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

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

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes

Specific details on test material used for the study:

Batch SP04027/GLP
stored at ambient temperature

Analytical monitoring:

no

Vehicle:

yes

Remarks:

de-ionised water

Details on test solutions:

A nominal 500 mg/l stock solution of ceftazidime dihydrochloride was prepared in
deionised water. Following shaking this stock was observed to be a clear colourless
solution. The initial pH of 3.42 was adjusted to 9.59 with 2M sodium hydroxide. Test
solutions were prepared by the addition of a known volume of this stock solution to a
total volume of 500 ml aerated dechlorinated water, synthetic sewage and activated
sludge as described in the experimental design (Table 1 ).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from Buckland Sewage Treatment Works, Newton
Abbot, Devon, UK, 1 day prior to the exposure of test and reference substances. This
works treats sewage of predominantly domestic origin .
On return to the laboratory, the activated sludge was settled and the concentrated sludge
was washed with OECD mineral salts medium. The washed settled sludge was fed with
50 ml of OECD synthetic sewage feed per litre of sludge per day and aerated at room
temperature, until it was used in the test.
The total filterable solids concentration was determined on the day of the test and was
found to be 5223 mg/l. The pH was adjusted to 7.08.

Synthetic sewage
A synthetic sewage mixture described by the OECD guideline was prepared,
containing the following constituents per litre of deionised water: 15.2 g of peptone,
10.5 g of meat extract, 2.9 g of urea, 0.7 g of NaCl, 0.4 g of CaCl2.2H20, 0.2 g of
MgSo4.7H20 and 2.8 g of K2HP04 .

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 +- 2 C

pH:

7.03 - 7.82

Dissolved oxygen:

6.5 mg O2/l

Salinity:

not applicable

Conductivity:

not measured

Nominal and measured concentrations:

nominal concentration of 100 mg/L

Details on test conditions:

Two replicate flasks of a single nominal 100 mg/l concentration of ceftazidime dihydrochloride were prepared, together with two control culture flasks. Five flasks containing the reference substance, 3,5-dichlorophenol, at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were also prepared. The experimental design is shown in
Table 1.
Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg r', an appropriate quantity of ceftazidime dihydrochloride, or 3,5 dichlorophenol stock solution and aerated tapwater to give a final flask contents volume of 500 ml. The exact quantities of each of these constituents are given in Table 1. The pH of each flask was measured before the start and at the end of the test.
Flasks were set up at 10 minute intervals and aerated at 20 ± 2°C for 3 hours. The temperatures of the flask contents were measured at the end of the 3 hours aeration using a mercury-in-glass thermometer.

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Mean control respiration rate = 31.1 mg 02/l/hr

Results with reference substance (positive control):

The reference substance, 3,5-dichlorophenol, caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained, the 3 hour EC50 value was estimated to be 9.5 mg/l (Fig I). This is within the expected normal range of 5 to
30 mg/l , indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other. Therefore, the mean control respiration rate was used in the calculation of the percentage inhibition.

Inhibition Results

Test Substance	Nominal test concentration (mg/l)	pH start	pH end	Respiration Rate mg O2/l/hr	Percentage mean control respiration rate	Percentage inhibition
Control	-	7.27	7.97	31.5	-	-
3,5-DCP	100	7.16	7.95	4.0	13	87
3,5-DCP	32	7.21	7.98	5.5	18	82
3,5-DCP	10	7.27	7.95	15.0	48	52
3,5-DCP	3.2	7.29	8.00	28.0	90	10
3,5-DCP	1.0	7.24	7.97	29.4	94	<10
Ceftazidime dihydrochloride	100	7.03	7.82	28.2	91	<10
Ceftazidime dihydrochloride	100	7.17	7.81	26.0	84	16
control	-	7.20	7.79	30.6	-	-

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for activated sludge respiration inhibition by the test substance is >100mg/l (nomnal concentration). Based on the results obtained the NOEC is <100mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L