2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

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

Study period:

2 May 2002 to 14 May 2002

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
A 2.00 mg/mL stock solution of the test material was prepared by weighing 4233.0 mg of test material, correcting for purity, and diluting in 2000 mL of reagent water in a Class A volumetric flask. Aliquots of the stock solution were used to add test material to the test flasks to achieve testing concentrations of 100, 250, 500, 750 and 1000 mg/L.

A stock solution of the reference material was prepared by weighing 501.6 mg, correcting for purity of 99.7 %, and diluting in 10 mL of 1 N NaOH and 20 mL of reagent water. Sulphuric acid was added until the 3,5-dichlorophenol reached the point of incipient precipitation (10 mL of 1 N H₂SO₄). The solution was then diluted to a final volume of 1000 mL using reagent water. The final concentration was 500 mg/L and the pH was 7.87. This stock solution was used to add reference material to the appropriate flasks. The reference material stock solution was stored under refrigeration.

Test organisms (species):

activated sludge

Details on inoculum:

MICROBIAL INOCULUM
The microbial inoculum used in the test was activated sludge collected on 13 May 2002, from aeration basin #1 at the Columbia Wastewater Treatment Plant in Columbia, Missouri. Approximately 20 litres of activated sludge were collected.

PREPARATION OF THE MICROBIAL INOCULUM
The microbial inoculum was prepared as follows. The suspended solids in the activated sludge were allowed to settle and the supernatant was siphoned until the volume of sludge was reduced from 20 to 4 L. Twelve 250-mL centrifuge bottles were filled with activated sludge and centrifuged at ~3000 rpm for ~5 minutes using an IEC CR-6000 centrifuge. The supernatant was discarded, six of the bottles were filled with activated sludge, and the bottles were centrifuged a second time. The supernatant was again discarded. After the supernatant was discarded, the activated sludge was rinsed by adding well water, shaking, and then re-centrifuging. The supernatant was discarded and the activated sludge was rinsed twice more in the same manner. The final supernatant was discarded. A suspension of the sludge pellets was prepared by adding ~200 mL of well water to each bottle. The contents of the bottles were combined in a 20-L carboy.
The combined sludge volume of 2680 mL was placed in an environmental chamber maintained at 20 ± 2 °C and aerated using air. Synthetic sewage feed was added to the sludge each day at approximately 50 mL/L to sustain it until the day of use.
The suspended solids concentration of the final activated sludge solution used for the test was 3.9 g/L.

COMPOSITION OF SYNTHETIC SEWAGE FEED SOLUTION
Synthetic sewage feed was made by dissolving the following nominal amounts of substances per 1 L of well water:
16 g peptone, 11 g beef extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl₂.2H₂O, 0.2 g MgSO₄.7H₂O, 2.8 g K₂HPO₄.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Test temperature:

20.2 - 20.6 °C

pH:

8.07 - 8.09 (control solutions); 7.11 - 8.14 (test material treatments); 3.61 (abiotic control)

Nominal and measured concentrations:

100, 250, 500, 750 and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000-mL glass flask
- Fill volume: 500 mL
- Aeration: Each contact flask was aerated in a temperature-controlled environmental chamber at 20 ± 2 °C for ~30 minutes.
- No. of vessels per test material concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per abiotic control (replicates): 1
- No. of vessels per reference material concentration (replicates): 1

CONTACT FLASK PREPARATION
Each flask contained a Teflon-coated magnetic stir bar and was placed on an insulated magnetic stir plate. Control flasks contained the microbial inoculum, well water, and synthetic sewage feed but no test or reference material. Reference material flasks, containing the microbial inoculum, well water, and synthetic sewage feed, were dosed with 3,5-dichlorophenol at concentrations of 3.2, 10 and 32 mg/L. Test material flasks contained the microbial inoculum, well water, synthetic sewage feed, and test material at nominal concentrations of 100, 250, 500, 750, and 1000 mg/L. An abiotic control flask was prepared and contained well water, synthetic sewage feed, and test material at a nominal concentration of 1000 mg/L. No microbial inoculum was added to the abiotic control flask.
The flasks were prepared in two groups. The first group included the following six contact flasks: Control-1, Abiotic Control, Test 750 mg/L, Test 500 mg/L, Reference 10 mg/L and Reference 3.2 mg/L. The second group included the following five contact flasks: Control-2, Reference 32 mg/L, Test 100 mg/L, Test 1000 mg/L and Test 250 mg/L. After preparing a group of contact flasks, the contents of each flask were stirred and aerated at ~800-1000 mL/minute. The second group of contact flasks was then prepared and the contents of each flask were stirred and aerated in the same manner.

TEST WATER
- Well Water: Well water (non-chlorinated) was used for all inoculum preparation procedures, synthetic sewage feed preparation and test flask preparations. The water was used directly from the faucet in the laboratory with no additional preparation.
- Reagent Water: Reagent water was purified, deionised and filtered using a Millipore Milli-Q Water Purification System. The filtrate was > 10 megohm.cm in resistivity, which is equivalent to or better than the ASTM Type II water resistivity requirement.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED
After a contact period of 30 minutes measurements for dissolved oxygen were taken with an ASR system (Strathkelvin Instruments ASR system). The ASR system provided the oxygen depletion curve as a plot of the dissolved oxygen concentration (mg O₂/L) versus time (seconds).

TEST CONCENTRATIONS
- Range finding study
A range-finding test was conducted on 2 May 2002. Although the EC50 for the reference material was acceptable (29.8 mg/L), the EC50 for the reference material in a second test performed on the same day was not in the acceptable range of 5 to 30 mg/L. The range-finding test was then repeated for the test and reference materials on 14 May 2002. Based on the results from the range-finding test, a definitive test was not conducted.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Percent inhibition values for test material treatments in the range-finding test were 0, 8, 9, 17, and 21 % for the 100, 250, 500, 750, and 1000 mg/L treatments, respectively. Based on these results, an EC50 value for the test material could not be determined but was > 1000 mg/L, the highest concentration tested, and a concentration that exceeds the test concentration of biodegradation studies. Therefore, a definitive test was not performed.

Results with reference substance (positive control):

The three reference substance flasks exhibited percent inhibition values of 15, 44, and 76 % at 3,5-dichlorophenol concentrations of 3.2, 10, and 32 mg/L, respectively. An EC50 value of 12.3 mg/L with 95 % confidence limits of 9.3 and 16.4 mg/L was calculated for 3,5-dichlorophenol using the Spearman-Karber method.

Reported statistics and error estimates:

The median effective concentration (EC50) for the reference material was statistically calculated using an EC50 SAS Program. The program calculates the EC50 statistic and its 95 % confidence limits using the Spearman-Karber method. The variables were calculated concentrations and percent inhibition at the corresponding concentration.

Water Quality Parameters

Temperature of the environmental chamber ranged from 20.2 to 20.6 °C during the range-finding test and during storage of the microbial inoculum prior to test initiation, which was within the range of 20 ± 2 °C as required by the test. The pH after the 30-minute contact period was 8.07 and 8.09 for the control solutions. The pH ranged from 7.11 to 8.14 for test material treatments with activated sludge. The pH of the reference substance treatments ranged from 8.11 to 8.22. The pH of the activated sludge solutions was within the acceptable range of 6.0 to 8.5. The pH of the abiotic control solution, which contained no activated sludge, was 3.61. The low pH of the abiotic control indicated that the activated sludge buffered the pH of the test material treatments with activated sludge.

Validity criteria fulfilled:

yes

Conclusions:

Inhibition of respiration by the test material was ≤ 21 % at the five concentrations tested (100 to 1000 mg/L). As respiration rate reduction was <50 % in all test material treatments, an EC50 value could not be calculated and is therefore reported as > 1000 mg/L, the highest concentration tested. The EC50 value for the reference material, 3,5-dichlorophenol, was determined to be 12.3 mg/L and was within the OECD 209 guideline range of 5 to 30 mg/L.

Executive summary:

The toxicity of the test material to microorganisms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209.

The respiration rate of an activated sludge inoculum in a synthetic sewage suspension, after aerating for 30 minutes in the presence of test material, was compared to the respiration rate of an activated sludge inoculum in a synthetic sewage suspension to which no test material was added. A range-finding test was conducted at nominal test material concentrations of 100, 250, 500, 750, and 1000 mg/L. Two control flasks containing an activated sludge and synthetic feed mixture with no test material added were also tested. An abiotic control was dosed at 1000 mg/L test material during the range-finding test and was used to measure chemical oxygen uptake. No inoculum was added to this flask. A reference substance, 3,5-dichlorophenol, a known microbial inhibitor, was tested to verify normal sensitivity of the microbial population. Reference substance concentrations of 3.2, 10, and 32 mg/L were tested during the range-finding test.

The two control flasks containing an activated sludge and synthetic feed mixture with no test material exhibited respiration rates within 15 % of each other as required by the OECD Guidelines, Section 209. Exposure of the activated sludge and synthetic feed mixture to the reference substance resulted in percent inhibition values of 15, 44 and 76 % for the 3.2, 10, and 32 mg/L treatments, respectively. An EC50 value of 12.3 mg/L was calculated for the reference substance and was within the acceptable range of 5 to 30 mg/L as specified in the OECD Guidelines, Section 209. The respiration rate of the abiotic control, 2.5 mg O₂/L/hr indicated that oxygen uptake by the test material was 3 % of the mean respiration rate of the biotic controls, 81.7 mg O₂/L/hr.

Inhibition of the respiration rate was 0, 8, 9, 17, and 21% for the 100, 250, 500, 750, and 1000 mg/L treatments, respectively. Because respiration rate reduction was <50 % in all test material treatments, an EC50 value could not be calculated and was estimated to be >1000 mg/L, the highest concentration tested. A definitive test was not conducted since the inhibition at the highest concentration tested was <50 %.

Description of key information

EC50 > 1000 mg/L, OECD 209, Heim & Heim (2002)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The toxicity of the test material to microorganisms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209. The study was awarded a reliability score of 1in line with the criteria of Klimisch et al. (1997).

The respiration rate of an activated sludge inoculum in a synthetic sewage suspension, after aerating for 30 minutes in the presence of test material, was compared to the respiration rate of an activated sludge inoculum in a synthetic sewage suspension to which no test material was added. A range-finding test was conducted at nominal test material concentrations of 100, 250, 500, 750, and 1000 mg/L. Two control flasks containing an activated sludge and synthetic feed mixture with no test material added were also tested. An abiotic control was dosed at 1000 mg/L test material during the range-finding test and was used to measure chemical oxygen uptake. No inoculum was added to this flask. A reference substance, 3,5-dichlorophenol, a known microbial inhibitor, was tested to verify normal sensitivity of the microbial population. Reference substance concentrations of 3.2, 10, and 32 mg/L were tested during the range-finding test.

The two control flasks containing an activated sludge and synthetic feed mixture with no test material exhibited respiration rates within 15 % of each other as required by the OECD Guidelines, Section 209. Exposure of the activated sludge and synthetic feed mixture to the reference substance resulted in percent inhibition values of 15, 44 and 76 % for the 3.2, 10, and 32 mg/L treatments, respectively. An EC50 value of 12.3 mg/L was calculated for the reference substance and was within the acceptable range of 5 to 30 mg/L as specified in the OECD Guidelines, Section 209. The respiration rate of the abiotic control, 2.5 mg O₂/L/hr indicated that oxygen uptake by the test material was 3 % of the mean respiration rate of the biotic controls, 81.7 mg O₂/L/hr.

Inhibition of the respiration rate was 0, 8, 9, 17, and 21% for the 100, 250, 500, 750, and 1000 mg/L treatments, respectively. Because respiration rate reduction was <50 % in all test material treatments, an EC50 value could not be calculated and was estimated to be >1000 mg/L, the highest concentration tested. A definitive test was not conducted since the inhibition at the highest concentration tested was <50 %.