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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
no guideline followed
Principles of method if other than guideline:
Tetrahymena pyriformis (in phosphate buffer) were subjected to sodium azide. Respiration, movement and phagocytosis were monitored.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
phosphate buffer, pH 6.0
Test organisms (species):
Tetrahymena pyriformis
Test type:
static
Limit test:
no
Total exposure duration:
1.25 h
Remarks on exposure duration:
Sodium azide was added to the phosphate buffer and observations were made in the subsequent 1h 15 minutes.
pH:
6.0
Nominal and measured concentrations:
0.01, 0.02, 0.05, 0.1, 0.5-5 mM, 10 mM
Reference substance (positive control):
no
Duration:
1.25 h
Dose descriptor:
NOEC
Effect conc.:
10 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: phagocytosis
Duration:
1.25 h
Dose descriptor:
NOEC
Effect conc.:
10 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
1.25 h
Dose descriptor:
NOEC
Effect conc.:
100 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cilia movement
Duration:
1.25 h
Dose descriptor:
LOEC
Effect conc.:
50 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: phagocytosis
Duration:
1.25 h
Dose descriptor:
LOEC
Effect conc.:
20 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
At the highest concentration tested (10 mM) sodium azide was markedly toxic to Tetrahymena, with inhibition of cilia movement and of phagocytosis of ink. At first, respiration increased but subsequently decreased after 2h of azide treatment. A markedly increase in oxygen consumption was observed at lower azide concentrations (0.5-5 mM); 0.02 mM still caused a slight promotion whilst with 0.01 mM, no effect on respiration was visible any more. Similar to the experiments with cyanide cilia movement and phagocytosis ability were hindered at the stated concentrations despite oxygen consumption increased. Cilia movement normalized at 0.1 mM, phagocytosis ability restarted at 0.05 mM and normalized not until 0.01 mM, which is a concentration that didn't affect respiration any more.

Table 1: Respiration, movement and phagocytosis ability of Tetrahymena after treatment with 0.1 mM sodium azide.

 

Sodium azide (0.1 mM)

N a

6; 2

Control QO2 ± SE b

18.4 ± 3.27

Experiment QO2 ± SE

40.0 ± 7.1

Exp. in % of control.

217

p% c

< 1

Control QCO2 ± SE

6.5

Experiment QCO2 ± SE

19.5

Exp. in % of control.

300

p%

---

Movement

Slightly impaired

Phagocytosis

Highly impaired

anumber of experiments

bSE: standard error

csignificance was determined using the method for paired observations

 

 

Validity criteria fulfilled:
not applicable
Conclusions:
The NOEC of sodium azide on the toxicity towards Tetrhymena pyriformis was 10 µM (equalling 680 µg/L). At 20 µM, an increased reespiration rate was observed, causing this concentration to be the LOEL in this experiment.
Executive summary:

Burmeister studied the toxicity of sodium azide and other chemicals against the ciliate Tetrahymena pyriformis. Studied parameters were respiration, movement, and phygocytosis ability of the ciliate. The measurements were conducted in a so called "Warburg-Gerät" (Warburg apparatus) which is a manometric oxygen consumption analyzer. The phagocytosis abillity was measured by the addition of ink and by addition of dead bacteria (Staphylococcus aureus) to the Tetrahymena culture. No further details on methods are given. The procedure is not performed according to a test guideline or GLP.

A slight increase in oxygen consumption was measured after incubation with 20 µM sodium azid which was more severe at higher concentrations. Phagocytosis abillity was hindered at concentrations of 50 µM and more and cilia movement was impaired at concentrations >100 µM. No alterations in all three markers were observed at concentrations of 10 µM sodium azide.

For this reason, the NOEL of sodium azide to Tetrahymena pyriformis was 10 µM and the LOEL was 20 µM because the impaired respiration rate at incubation with this concentration.

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
no guideline followed
Version / remarks:
No guideline available at the time of study performance.
Principles of method if other than guideline:
Guideline not followed. The study included two different experiments, the first investigated the effect of sodium azide on the microbial growth and respiration of 92 bacterial strains, in single inoculum experiments. The second investigated the effect sodium azide in aerated solutions on the growth of particular bacteria, Aerobacter aerogenes.
GLP compliance:
no
Specific details on test material used for the study:
No data.
Analytical monitoring:
not required
Vehicle:
not specified
Details on test solutions:
- Test solutions were sterile
-Test solutuions were prepared at 0.01, 0.02 and 0.03 %, calculated to be equivalent to 0.1, 0.2 and 0.3 mg/L.
Test organisms (species):
other: pure cultures of 92 bacterial strains
Details on inoculum:
- 92 bacterial strains were selected.
- The strains were largely obtained from stock cultures held by the Hygienic Laboratory.
Test type:
static
Water media type:
other: Infusion agar or solid agar
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Not stated
Test temperature:
- plates and tubes incubated at 37 °C
pH:
Beef infusion broth at 7.4
Details on test conditions:
Experiment 1: Measurement of the growth of selected strains of bacteria at various concentrations of Sodium azide after 48 hours
- Test solutuions were prepared at 0.01, 0.02 and 0.03 %, calculated to be equivalent to 0.1, 0.2 and 0.3 mg/L.
- The basic medium for cultural studies was beef infusion broth (pH 7.4).
- The sodium azide was added to the infusion broth, and solutions were sterilized.
- The sterile media were poured into the Petri dishes and allowed to solidify. The petri dishes were then inoculated with the particular bacterial strain by streaking with a loopful of an 18-24 hour broth culture.
- Infusion broths containing similar amounts of the chemical was also inoculated.
- All plates and tubes of agar or broth were incubated. Variable conditions were used for the optimum growth of different strains.

Experiment 2: the effect of aeration on the growth of Pseudomonas aeruginosa in the prescence of Sodium azide in broth, 48 hours, 37 °C
- The effect of aeration on P. aeruginosa exposed to Sodium azide was studied due to an apparent resistance observed in Experiment 1, which was unlike most other gram negative bacteria.
- Two strains of P. aeruginosa were tested, the S strain (did not produce pyocyanine) and strain 2 (produced pyocyanine).
- Ten ml of infusion broth (with and without sodium azide) was placed in 50 ml Erlenmeyer flasks. The media were inoculated with bacteria and incubated ay 37 °C. Flasks were aerated in 3 different ways.
Reference substance (positive control):
not specified
Details on results:
Experiment 1 (see tables 1, 2 and 3): Measurement of the growth of selected strains of bacteria at various concentrations of Sodium azide after 48 hours:
- The data indicates that Sodium azide exerted a marked bacteriostatic action against gram-negative bacteria as a group. Of the 41 strains tested on infusion agar containing Sodium azide, only 24 grew at a concentration of 0.01 %, 6 grew at a concentration of 0.02 %, while all but Pseudomonas aeruginosa failed to grow in the presence of 0.03 %. On several occasions, strains of P. aeruginosa freshly isolated from clinical material grew as well on media containing 0.03 % Sodium azide as on plain infusion agar. The results with broth containing Sodium azide were essentially the same, except that P. aeruginosa and Pseudomonas fluorescens, which were more resistant to the chemical in infusion agar than the other gram negative organisms, were very susceptible under these conditions.
- Fifty-one strains of gram-positive bacteria were similarly tested on the solid media. The data given in table 3 show that Sodium azide in the concentrations employed had little or no effect on the aerobic growth of streptococci, pneumococci or lactobacilli. The anaerobes were likewise not inhibited. The aerobic sporulating rods, and Staphylococcus citreus were very susceptible to the chemical, while Staphylococcus aureus and Staphylococcus albus were moderately resistant. The hemolysis produced by Streptococcus viridans and the pneumococcus was characterized by a large brownish zone around the colony, as contrasted to the usual small area of greening around the colonies in the absence of the chemical.

Experiment 2 (see table 4): the effect of aeration on the growth of P. aeruginosa in the presence of Sodium azide in broth, 48 hours, 37 °C
- The data presented in table 4 indicates that all three methods of aeration of Sodium azide broth cultures of a pyocyanine-producing strain of P. aeruginosa increased the growth of the organism to a marked degree. On the other hand, aeration had little or no effect on the proliferation of the strain which produced no pyocyanine.

Table 1. Growth of gram negative bacteria on infusion agar containing various concentrations of Sodium azide, 48 hours, 37 °C

Organisms

Number of Strains Tested

Concentrations of NaNs (percent)

0.01

0.02

0.03

0.00

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

E. Coli

3

2

+

0

-

0

-

3

+++

E.typhosa

5

2

++

1

+

0

-

5

+++

A.aerogenes

2

0

-

0

-

0

-

2

+++

P. vulgaris

7

7

+ (NS)

0

-

0

-

7

+++ (S)

S. dysenteriae

3

2

+

0

-

0

-

3

+++

Salmonella

8

5

++

1

+

0

-

8

+++

P. aeruginosa

3

3

+++

3

++

3

+

3

+++

P. fluorescens

1

1

++

1

++

0

-

1

+++

C. violaceum

2

0

-

0

-

0

-

2

+++

H. ducreyi*

1

1

+

0

-

0

-

1

+++

Flavobacterium

2

0

-

0

-

0

-

2

+++

Gonococcus*

2

1

+

0

-

0

-

2

+++

Meningococcus*

2

0

-

0

-

0

-

2

+++

+, poor growth; ++, good growth; +++, control growth; -, no growth.

(S) spreading. (NS) no spreading.

* Blood agar base used.

Table 2. Growth of gram-negative bacteria in broth containing various concentrations of sodium azide, 48 hours, 370C.

Organisms

CONCENTRATIONS OF SODIUM AZIDE (PER CENT)

 

0.01

0.02

0.03

0.05

0.00

P. vulgaris (7)

++

+

-

-

+++

S. dysenteriae (3)

+

+

+

-

+++

E. typhosa (5)

+

+

-

-

+++

E. coli (3)

++

+

+

-

+++

A. aerogenes (2)

+

-

-

-

+++

P. aeruginosa (3)

++

-

-

-

+++

P. fluorescens (1)

+

-

-

-

+++

+, poor growth; ++, good growth; +++, control growth;-, no growth. ( ) number of strains tested.

Table 3. growth of gram positive bacteria on infusion agar containing various concentrations of sodium azide, 48 hours, 37 °C

Organisms

Number of Strains Tested

Concentrations of NaNs (percent)

0.01

0.02

0.03

0.00 (control)

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

Number of strains giving growth

Amount of growth

S. hemolyticus

18

16

+++

16

+++

15

+++

18

+++

 

 

2

++

2

++

3

++

 

 

S. viridans

7

7

+++

7

+++

7

+++

7

+++

S. anhemolyticus

3

3

+++

3

+++

3

+++

3

+++

Pneumococcus

3

3

+++

3

+++

3

+++

3

+++

S. aureus

3

3

++

3

++

3

+

3

+++

S. albus

2

2

++

2

+

2

+

2

+++

S. citreus

1

0

-

0

-

0

-

1

+++

C. diphtheriae

2

2

+++

2

++

2

+

2

+++

B. anthracis

1

0

-

0

-

0

-

1

+++

B. subtilis

2

0

-

0

-

0

-

2

+++

B. mesentericus

1

1

++

0

-

0

-

1

+++

B. mgatherium

1

0

-

0

-

0

-

1

+++

Lactobacillus

2

2

+++

2

+++

2

++

2

+++

C. welchii

1

1

+++

1

+++

1

+++

1

+++

C. novyi

1

1

+++

1

++

1

++

1

+++

C. sporogenes

1

1

+++

1

+++

1

+++

1

+++

C. tetani

1

1

+++

1

+++

1

+++

1

+++

C. botulinum

1

1

+++

1

+++

1

+++

1

+++

+, poor growth; ++, good growth; +++, control growth; -, no growth.

Table 4. Effect of aeration on the growth of P. aeruginosa in the presence of sodium azide in broth, 48 hours, 37C.

Type of Aeration

Concentration of Sodium Azide

(Percent)

Growth of strain S*

Growth of strain 2*

Media in culture tubes (surface volume ratio = 0.15)**

0.00

+++

+++

0.01

++

++

0.02

-

+

0.03

-

-

Media in flasks (surface volume ratio = 1.6)**

0.00

+++

+++

0.01

++

++

0.02

-

++

0.03

-

+

Media in flasks (aerated by mechanical shaking)

0.00

+++

+++

0.01

++

+++

0.02

-

+++

0.03

-

++

Media in flasks (aerated by passage of air through media)

0.00

+++

+++

0.01

++

+++

0.02

+

+++

0.03

-

++

+, poor growth; ++, good growth; +++, control

* Strain S produces no pyocyanine.

Strain 2 produces pyocyanine.

** surface area in sq. mm.

volume in ml.

Validity criteria fulfilled:
not specified
Conclusions:
Sodium azide was shown to inhibit the growth of many different species of bacteria at a concentration of 0.1 mg/L. The extent of inhibition was affected by the concentration of sodium azide, aeration, and the species of bacteria. Some species demonstrated greater resistance than other species.
Executive summary:

A study was conducted to determine the effect of Sodium azide on the growth of 92 different species of bacteria, investigating the influence of bacterial strain, concentration of Sodium azide, and various environmental conditions. The main conclusions were:

1. Sodium azide in concentrations of 0.01 to 0.03 % (0.1 to 0.3 mg/L) in infusion agar was bacteriostatic for gram-negative bacteria. Of the 41 strains tested, only 3 strains of Pseudomonas aeruginosa grew in the presence of 0.03 % (0.3 mg/L) of the chemical.

2. Streptococci, pneumococci, anaerobes, and lactobacilli were resistant to 0.03 % (0.3 mg/L) Sodium azide. However, the gram-positive spore-forming aerobes, and Staphylococcus citreus were very sensitive, while Staphylococcus albus, Staphylococcus aureus, and Corynebacterium diphtheriae were moderately resistant.

3. Results using broth containing Sodium azide were essentially the same as those obtained with solid media. An exception was P. aeruginosa which was far more sensitive in Sodium azide broth.

4. Twelve strains of facultative anaerobes were cultured under anaerobic conditions on infusion agar containing 0.01 to 0.03 % (0.1 to 0.3 mg/L) Sodium azide. The results were essentially the same as those obtained under aerobic conditions, with the exception that S. albus, S. aureus, and P. aeruginosa were more sensitive to the chemical when cultured in the absence of free oxygen.

5. Aeration of Sodium azide broth cultures of a pyocyanine-producing strain of P. aeruginosa increased the growth of the organism in the presence of the chemical.

The test system is only capable of determining effects on growth of bacterial cultures, but not on the parameters of interest (e.g. respiration or similar vital functions). No conclusions can be drawn regarding inhibition of microbial communities e.g. in wastewater treatment plants. A LC50 and/or an NOEC cannot be determined on the basis of the presented data.

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
see materials and methods, and executive summary
Principles of method if other than guideline:
The principle of the test method was to isolate streptococci bacteria from bacterial mixtures containing spreading strains of Bacterium proteus, based on the known inhibitory nature of sodium azide specifically on gram negative bacteria. Once an inhibitory concentration of Sodium azide was determined, the inhibitory nature of Sodium azide was analysed on 7 urine and faeces samples.
GLP compliance:
not specified
Specific details on test material used for the study:
No additional details provided
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- aqueous solutions of Sodium azide were used
Test organisms (species):
other: Various strains of bacteria
Test type:
static
Water media type:
other: agar plate
Limit test:
no
Nominal and measured concentrations:
Nominal concentrations - 0.008, 0.010, 0.05, 0.10, 0.25 and 0.35 % (0.08, 0.1, 0.5, 1.0, 2.5, 3.5 mg/L)
Details on test conditions:
- Sodium azide was added at variable concentrations to nutrient agar.
- The agar was autoclaved and either 5 % sheep or rabbit blood added.
- The first part of the experiment was to determine the concentration which would inhibit growth of Bacterium proteus.
- Once this concentration was established (0.01 %), 7 specimens of urine and feces were streaked on the agar with 0.01 % concentration Sodium azide and cultured.
- No further details on the methodology and test conditions were provided
Reference substance (positive control):
no
Key result
Dose descriptor:
IC100
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: Bact. proteus
Key result
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: bacterial populations
Details on results:
Table 1 details the results on the growth and inhibition of bacterial populations at different concentrations of Sodium azide. At 0.008 % sodium azide (equivalent to 0.08 mg/L) growth of a range of bacterial types was comparable to the control, with no Sodium azide present. At concentrations of 0.1 mg/L, growth was affected, with greater growth inhibition at higher concentrations. At 0.1 % (equivalent to 1 mg/L) sodium azide was shown to be an excellent inhibitor of Bacterium proteus.

Table 1. Concentration of sodium azide necessary to permit growth of Streptococci and inhibit the spreading of Bacterium proteus

Concentration Sodium azide (%)

Bact. Proteus 2

Bact. Proteus 2792N

Bact. coli.

Bact. typhosum

Str. hemolyticus

Str. fecalis

Str. viridans

Group A (9)

Group B

Group D

Group G

0.35

0

0

+

0

0

0

0

0

 

 

0.25

0

0

0

0

0

0

 

 

0.10

0

0

0

0

0

0

 

 

0.05

0

0

++

±

±

±

+

+

0.010

 

++

NS

++

NS

++

++

+++

+++

+++

+++

0.008

 

+++

S

+++

S

+++

+++

+++

+++

+++

+++

Control

 

+++

S

+++

S

+++

+++

+++

+++

+++

+++

NS – No Spreading

S - Spreading

Validity criteria fulfilled:
not applicable
Conclusions:
At 0.1 % (equivalent to 1 mg/L) sodium azide was shown to be an excellent inhibitor of Bact. proteus.
Executive summary:

A study was conducted to isolate streptococci bacteria from bacterial mixtures containing spreading strains of Bacterium proteus, based on the known inhibitory nature of Sodium azide specifically on gram negative bacteria. Once an inhibitory concentration of Sodium azide was determined, the inhibitory nature of Sodium azide on bacteria in 7 urine and feces samples was studied. A concentration of 0.1 % (equivalent to 1 mg/L) was required for inhibition of Bact. proteus, such that gram positive bacteria could be grown. Sodium azide was shown to be effective at inhibiting the growth of different types of bacteria, with the level of inhibition related to the concentration of Sodium azide. An LC100 of 1 mg/L for Bact. proteus has been derived from the data. However, the test system is only capable of determining effects on growth of bacterial cultures, but not on the parameters of interest (e.g. respiration or similar vital functions). No conclusions can be drawn regarding inhibition of microbial communities e.g. in wastewater treatment plants. A LC50cannot be determined on the basis of the presented data. Though no statistical analysis was conducted, no effect on the growth of microorganisms was observed at a concentration of 0.8 mg/L, hence, presumably represents a NOEC for Bact. Proteus.

Description of key information

As key study a GLP conform study following the OECD guideline 209 was chosen (Maischak 2017). The most sensitive effect concentration were derived for the endpoint of inhibition of nitrification:

NOEC = 0.03 mg/L

EC50 = 5.60 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
5.6 mg/L
EC10 or NOEC for microorganisms:
0.03 mg/L

Additional information

The toxicity towards microorganisms was assessed, evaluating four studies: one study according to the OECD guideline 209 and GLP conform (Maischak 2017) and three supporting studies (Burmeister 1974, Lichstein 1943, Snyder 1940). Two of the supporting studies were rated with Klimisch 3 due to unsuitable test systems. One study was rated with Klimisch 2 which exposed Tetrahymena pyriformis to sodium azide.The key study, performed according to OECD test guideline 209 (Maischak, 2017), investigated the effect of sodium azide on total respiration, heterotrophic respiration, and nitrification of microorganisms from a municipal sewage treatment plant. The most sensitive parameter was nitrification, with an EC50 of 5.6 mg/L and a NOEC of 0.03 mg/L.