Trisodium [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]manganate(3-)

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Standard OECD Guideline study, Conducted according to GLP.

Justification for type of information:

A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment.
The source substance is a chelating agent in a target substance. The only difference between the target and the main source substance is presence of manganese (Mn) cation instead two H+ cations.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid)

EC Number:

200-652-8

EC Name:

N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid)

Cas Number:

67-43-6

Molecular formula:

C14H23N3O10

IUPAC Name:

2-[bis[2-(bis(carboxymethyl)amino)ethyl]amino]acetic acid

Details on test material:

Test Material: diethylenetriaminepentaacetic acid
Supplier, City and State (Lot #): K. Svatek, Versene® Chelants Quality/Analytical, The Dow Chemical Company, Freeport, Texas (Lot #QA2101D6F6).
Synonyms: DTPA
Physical Properties: White solid
Molecular Formula: C14H23N3O10
Molecular Weight: 393.347
Compound Purity: The sample has a reported purity of 99.41%

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The microbial inoculum consisted of activated sludge mixed liquor, collected from the Midland Municipal Wastewater Treatment Plant (Midland, Michigan). This facility treats an excess of 3.0 x 106 gallons of wastewater daily, of which > 90% is from domestic sources. The mixed liquor inoculum was collected the day before the test was initiated and was continuously aerated until used. Prior to inoculation of the mineral medium, the mixed liquor suspended solids (MLSS) concentration of the mixed liquor was determined. Based on this determination, the sterile mineral medium (neutral and alkaline pH) was inoculated with the activated sludge mixed liquor to yield a final MLSS concentration of 30 ± 1 mg/L. The pH of the inoculated neutral mineral medium was measured and, if necessary, was adjusted to 7.4 ± 0.2. The pH of the inoculated alkaline mineral medium was adjusted to within pH 8.5-9.0 using a concentrated NaOH solution. After two hours of aeration, the pH 7 reaction vessels were filled and dosing was initiated. The remaining inoculated mineral medium was adjusted to pH 8.86 using 5N NaOH and aerated for greater than one hour prior to dispensing into the pH 9 reaction vessels.

Chemicals and Reagents:
Deionized water used in preparation of the mineral medium, analytical reagents, and calibration standards was purified through a MilliQ® (Millipore Corporation, Bedford, Massachusetts) water treatment system. All other chemicals used were of reagent grade and purchased from commercial sources.

Mineral Medium:
The mineral medium specified by OECD Guideline 301F was prepared by dissolving appropriate volumes of concentrated mineral stock solutions in MilliQ® water. Two batches of mineral medium were prepared to evaluate biodegradation under neutral and alkaline pH conditions. After preparation, the medium was sterilized in an autoclave using a 30-minute liquid cycle (121°C/15 p.s.i.).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

54 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation 3

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The biodegradation reactions were contained in specially designed 1-liter reaction vessels, each containing a 500-ml portion of the inoculated mineral medium. A total of sixteen (16) reaction mixtures were prepared as described in Table 1. The test and reference materials were added to the appropriate reaction mixtures as concentrated aqueous solutions to give 54 mg/L test material and 100 mg/L Benzoate. Single killed control reactions containing 54 mg/L test material were prepared in chemically-sterilized mineral medium (250 mg/L HgCl2) to verify the absence of O2 consumption and/or CO2 evolution in the absence of biological activity.

After addition of test material, reference compound and chemical sterilant to the appropriate reaction vessels, the pH of each reaction mixture was measured. pH 7 reaction mixtures were adjusted to pH 7.4 ± 0.2 and pH 9 reaction mixtures were adjusted between pH 8.5 and pH 9 with 1N HCl or 1N NaOH as necessary. Twenty-five milliliter aliquots of the reaction mixtures from each vessel were removed to determine the initial concentrations of dissolved organic carbon (DOC), nitrate, and nitrite. After connection to the respirometer system, the reaction vessels were purged with ambient air, and the associated headspace volume of each individual reaction vessel was determined by the respirometer system. Other specific operating parameters for the respirometer system are described below.

No Positive Controls were prepared for the pH 9 sample set as a result of an error in dosing which created two additional Toxicity Controls. Viability of the inoculum was confirmed by the response of the pH 9 Toxicity Controls and correlating the results with the pH 7 Toxicity Control and the pH 7 Positive Controls. The pH 9 reaction vessels (Chambers 1-8) were configured to allow for in study sampling to monitor pH of the reaction mixtures. An in-line valve was placed on the vent line and another valve on the vent port of each reaction vessel cap. The cap was fitted with a special nut to accommodate a Lure Lock syringe adapter. A Teflon® sampling line 3-4” long was attached to the interior vent port of each cap which did not contact the reaction mixture. At each sampling interval, both valves were closed, sequestering the reaction mixture in the bottle, and in the vent line to the expansion units. A 10 mL disposable syringe with a luer Lock adapter was attached to the reaction vessel sampling port and the cap valve was opened. The bottle was then tilted enough so the sampling line was submerged in the reaction mixture. A 10 mL aliquot was removed and transferred to a 20 mL scintillation vial, which was used to measure the pH. Adjustments were made to the subsample such that the pH was within a range of pH 8.5-9 using 0.01
N NaOH. The volume of NaOH required to adjust the pH of the entire reaction mixtures within range was extrapolated from the subsample. The extrapolated volume was added to the 10 mL aliquot and returned to the reaction vessel. The mixture was allowed to mix for several minutes and the pH was monitored again. This process was repeated until a stable pH between 8.5-9 was obtained. The biodegradation reactions were incubated in a darkened room at a target temperature of approximately 22 ± 1.0°C. The reactions were magnetically stirred at 150 r.p.m. over the entire 28-day test period.

Frequency of Sampling
Measurements of gas phase O2 and CO2 in the reaction vessels occurred on six-hour sample intervals over the entire 28-day test period. Upon completion of day 28 gas measurements, the reaction mixtures in each vessel were sampled for final DOC, nitrite, and nitrate measurements. The pH of each reaction mixture was also determined at the end of the 28-day test. The pH 9 reaction mixtures were monitored for pH and adjusted weekly, as necessary, in an effort to maintain a pH between 8.5 to pH 9 for the term of the study.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Test performed as expected. The toxicity controls indicated that the test material was not inhibitory to the microbial innoculum, and the positive control confirmed the assay functioned as expected.

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

0

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: pH 7 reaction mix

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

0

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: pH 7 reaction mix

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

6.6

Sampling time:

28 d

Remarks on result:

other: pH 9 reaction mix

% Degradation 4

Parameter:

% degradation (CO2 evolution)

Value:

9.3

Sampling time:

28 d

Remarks on result:

other: pH 9 reaction mix

Details on results:

No biodegradation of the test material was observed. DTPA did not meet the criteria of a “readily biodegradable" classification according to the current guidelines of the OECD 301 F: Manometric Respirometry Test. Increasing the pH of the reaction mixtures from 7 to approximately 9 slightly increased biodegradation (approximately 7% based on O2 consumption).

BOD5 / COD results

Results with reference substance:

Biodegradation of benzoate based on O2 consumption exceeded 60% after only 1.9 days, and reached > 100% after 28 days.

Any other information on results incl. tables

None.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

No biodegradation of the test material was observed. DTPA did not meet the criteria of a “readily biodegradable" classification according to the current guidelines of the OECD 301 F: Manometric Respirometry Test. Increasing the pH of the reaction mixtures from 7 to approximately 9 slightly increased biodegradation (approximately 7% based on O2 consumption).

Executive summary:

The ready biodegradability of Diethylenetriaminepentaacetic acid (DTPA) was evaluated using the OECD Guideline 301 F: Manometric Respirometry Test. Biodegradation of the compound was determined at an initial concentration of 54 mg/L in pH 7 and pH 9 reaction mixtures containing a dilute municipal activated sludge inoculum. Oxygen consumption and CO2 evolution resulting from biodegradation of the compound were measured over 28 days using a Columbus Instruments MicroOxymax® respirometer system (Columbus Instruments, Inc., Columbus, Ohio). There was no degradation observed in the pH 7 matrix over the 28-day evaluation period while minimal degradation at pH 9 over the 28-day evaluation period was noted. These results demonstrate that the test material does not meet current OECD criteria for “ready biodegradability” in the Manometric Respirometry Test. Biodegradation as determined from oxygen consumption (compared to the chemical oxygen demand (COD) of the test material) is required to reach the required 60% level within a 10-day period following onset of biodegradation. Net oxygen consumption and mineralization of the test material at pH 7 were both negligible and 6.6 and 9.3%, respectively, after 28 days at pH 9, never reaching onset of biodegradation at either pH. This experiment does demonstrate that increased biodegradation of DTPA is a function of pH. The suitability of the test procedure and microbial inoculum was verified by rapid biodegradation of a positive control compound (sodium benzoate) in a pH 7 reaction mixture. Biodegradation of benzoate based on O2 consumption exceeded 60% after only 1.9 days, and reached > 100% after 28 days. Toxicity control reactions containing a mixture of 100 mg/L benzoate and 54 mg/L test material showed no evidence for inhibition of biodegradation at either pH. The observed O2 consumption and CO2 production in the biodegradation reactions can be attributed solely to biological activity, as no net O2 consumption or CO2 production was observed in biologically inhibited control reactions containing 54 mg/L of the test material.