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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 april 2017 to 26 october 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Modified extended OECD 301B (CO2 evolution test)
Deviations:
yes
Remarks:
pH of the abiotic control was not determined at the end of the study, sampling intervals were diffetent to study plan. These deviations are no impact on the study
Principles of method if other than guideline:
The procedures followed during this study are detailed in the protocol entitled "Rhodiantal Original IBCH - Determination of the Biodegradability of a Test Substance Based on a Modified EXTENDED OECD Method 301B (CO2 Evolution Test)" . The methods described in this protocol meet the standard procedures described in the Organization for the Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 301B, Ready Biodegradability (OECD, 1992) and those published in the Official Journal of the European Communities, C.4-C (EC, 1992).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Concentrations were not adjusted for the purity of the test substance. The percent carbon was calculated to be 81.29% based on the chemical formula (C16H28O).
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, non-adapted
Details on inoculum:
The activated sludge used for this study was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste. Approximately 8 L of activated sludge was collected on 11 May 2017 and transported to Smithers Viscient. Upon arrival at Smithers Viscient, the sludge was passed through a 2-mm sieve and centrifuged at 1000 rpm for ten minutes. The supernatant was discarded, the sludge was washed with mineral medium (SMV No. 11May17L37-F) and the contents were centrifuged again and the supernatant was discarded. The moisture content of the activated sludge was determined, using a Sartorius MA-150 automated moisture analyzer, to be 95.05% and the percent solids was determined to be 4.95%. An inoculum solution with 15 mg suspended solids/mL was prepared (60.61 g wet weight sludge brought to 200 mL with mineral medium), stirred with a Teflon magnetic stir bar at 22 ± 2 °C, and aerated until used. The test suspension vessels, the blank vessels, the procedural control vessel, the toxicity control vessel, and the pH/TIC vessel all received 6 mL of the inoculum to produce an activated sludge concentration of 30 mg solids/L.
Duration of test (contact time):
56 d
Initial conc.:
10 mg/L
Based on:
ThCO2
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The aqueous medium for testing provided the essential nutrients, except for carbon, necessary to sustain the inoculum throughout the testing period.
Solutions were prepared using purified reagent grade water . The prepared mineral salts medium solutions 1 through 4 were prepared separately and kept refrigerated (approximately 2 to 8 °C) until use on day -1.

Phosphate buffer:
KH2PO4 : 8.50 g/L of stock; 10 ml of stock per L of medium
K2HPO4 : 21.75 g/L of stock
Na2HPO4 : 26.64 g/L of stock
NH4Cl : 0.50 g/L of stock
Calcium chloride: CaCl2: 27.50 g/L of stock; 1 ml of stock per L of medium
Magnesium sulfate: MgSO4·7H2O: 22.50 g/L of stock; 1 ml of stock per L of medium
Ferric chloride: FeCl3·6H2O: 0.25 g/L of stock; 1 ml of stock per L of medium
In order to avoid having to prepare this solution immediately prior to use, this stock solution was acidified with one drop of concentrated hydrochloric acid per liter of mineral media stock solution.
Salts with different amounts of hydration were able to be used and weights were adjusted proportionately. High purity reagent grade water, free from inhibitory concentrations of toxic substances (e.g., Cu2+ ions), was used for the preparation of the mineral medium and all dosing stock solutions. Only one batch of water, which had been checked by DOC analysis, was used. The water contained no more than 10% of the total carbon content introduced by Rhodiantal Original IBCH. The pH of the test medium was 7.4 and required no adjustment.

- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 22 +/- 2 °C
- pH: 7.46 - 7.56
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: no
- Suspended solids concentration: no
- Continuous darkness: yes

TEST SYSTEM
Each test unit consisted of a 4-L glass bottle with a rubber stopper into which one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were inserted. Prior to test initiation, the test vessels were acid washed and rinsed repeatedly with reagent grade water. The stainless steel needle was extended through the stopper into the test solution serving as a sampling port for solution samples. A rubber policeman cap was used to cover the top of the sample port. The glass tubing provided the inlet and outlet ports for air exchange. CO2-free air was pumped under positive pressure through a hydration flask before entering the test system. The outlet port of each system was connected to two CO2 effluent gas traps, the first consisting of 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH. The test vessels were identified with the study number, sample name, KOH trap number, and the sample ID number. Each test vessel was placed on a magnetic stir plate located in a dark environmental chamber set to maintain a temperature of 22 ± 2 °C.
Seven test vessels were established: two for Rhodiantal Original IBCH, two inoculum blanks, one sodium benzoate procedural control, one toxicity control, and one abiotic control. An additional vessel was established in the same manner as the test suspension vessels, but was used only for pH measurements and sampling for TIC and TC on day 0. This vessel was not attached to KOH traps and was disposed of after day 0.
On day -1, seven 4.0-L vessels were established by adding either 2991 mL of mineral medium (for the procedural and toxicity control) or 2994 mL of mineral medium (for the Rhodiantal Original IBCH test suspensions, the pH/TIC test vessel, and the inoculum blanks). A 6-mL aliquot of the activated sludge inoculum was added to each vessel for a total volume of 2997 mL or 3000 mL per vessel. An eighth vessel, the abiotic control vessel, only received 3000 mL of mineral media. These eight test vessels were attached to a CO2-free compressed air gas tank and aerated under positive pressure. The vessels were mixed and purged with CO2-free air until day 0 to remove any residual inorganic carbon in the test system prior to test initiation.
At test initiation (day 0), Rhodiantal Original IBCH replicate vessels (A and B) and additional sampling vessel were dosed. The total fortification was 10 mg C/L in the test suspension vessels.
The inoculum blank control vessels only received inoculum and mineral medium. The toxicity control vessel, which was fortified in the same manner as the test suspension vessels, also received 3.0 mL of the 10 mg C/mL sodium benzoate stock solution for a total fortification of 20 mg C/L (test and reference substance). The sodium benzoate procedural control was fortified with 3.0 mL of the sodium benzoate stock solution for a final concentration of 10 mg C/L. The pH test vessel was prepared in a similar manner to the test suspension vessels.

SAMPLING
All test systems were aerated continuously for 56 days under positive pressure using CO2-free air in order to provide oxygen for the microbes and to capture evolved carbon dioxide. The temperature of the environmental chamber was recorded daily throughout the exposure period using a digital minimum-maximum thermometer (VWR).
On test days 3, 7, 10, 14, 18, 21, 24, 28, 46, and 56, a 7-mL sample was removed from the first KOH carbon dioxide trap on each test system and analyzed for CO2 evolution. On day 56, 1 mL of concentrated hydrochloric acid (HCl) was added to each test vessel following pH measurements to terminate biological activity. Aeration was continued over the weekend to drive any residual inorganic carbon from the test vessels. After the acidification and aeration period, 7-mL samples for analysis of CO2 evolution were removed from the first and second traps. The amount of evolved CO2 in each trap was determined using a Shimadzu TOC V-CPH Carbon Analyzer.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes

STATISTICAL METHODS:
The Shimadzu TOC V-CPH Carbon Analyzer was used to analyze the samples removed from the KOH traps. TIC values were compared to a standard curve of at least five concentrations of sodium bicarbonate in order to determine the quantity of CO2 evolved.
The amount of carbon dioxide evolved from each test system was adjusted by subtracting the CO2 production value from the blank control. The percent biodegradability for each test system was calculated using the following equation and is expressed as cumulative percent biodegradation (or percent of theoretical CO2 production).

Reference substance:
benzoic acid, sodium salt
Remarks:
Lot Number BCBG3545V, CAS No. 532-32-1, with a reported purity of 99.9%, was received from Sigma Aldrich, Milwaukee, Wisconsin on 29 November 2012. Total organic carbon was calculated to be 58.23% based on the empirical formula (C7H5NaO2).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
56 d
Details on results:
The temperature in the environmental chamber was recorded daily, with the exception of weekends, throughout the study. The temperature ranged from 20 to 21 ºC. The pH of the test medium was measured to be 7.4 upon preparation. The pH of the pH check vessel on day 0 was measured to be 7.46. The pH values of the study vessels ranged from 7.47 to 7.56 at the end of the study on day 56.
The total inorganic carbon measured in the KOH traps was used to calculate the cumulative CO2 evolved from the test vessels. The mean cumulative CO2 values from the Rhodiantal Original IBCH and inoculum blank test vessels at day 56 were 29.36 and 38.67 mg/L, respectively. The cumulative CO2 values from the procedural and toxicity controls were 60.84 and 72.40 mg/L, respectively. The cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, values for the Rhodiantal Original IBCH test vessels, the procedural control, and the toxicity control were calculated to be -25.38, 60.48, and 45.99% respectively. The negative value (-25.38%) indicates that the CO2 evolution was less than the inoculum control. The cumulative net percent CO2 evolved from the procedural control was 66.12% of theoretical by day 14, thus meeting the “pass” criteria of the test (reaching 60% or greater CO2 evolution within 14 days). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.
The toxicity control produced a cumulative net CO2 percentage of 44.99% by day 14, demonstrating that Rhodiantal Original IBCH was not toxic to the inoculum as defined by OECD Guideline 301B (i.e., < 25% on day 14 is considered toxic).
The abiotic control produced cumulative CO2 percentages much less than the inoculum controls, as expected, and therefore showed no biodegradation.
Validity criteria fulfilled:
yes
Remarks:
see details on results
Interpretation of results:
not readily biodegradable
Conclusions:
Based on the extent of CO2 evolution during this study, Rhodiantal Original IBCH cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since it did not achieve 60% CO2 evolution within a 10-day window of reaching 10% biodegradation during the first 28 days of the study.
Executive summary:

This study was performed to determine the potential for ultimate biodegradation of Rhodiantal Original IBCH in water by the carbon dioxide evolution method following OECD Test Guideline 301B. The amount of carbon dioxide (CO2) released upon biodegradation of Rhodiantal Original IBCH and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. An abiotic control (containing Rhodiantal Original IBCH and sterilizing agent), two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance), and a toxicity control (containing inoculum, reference substance, and Rhodiantal Original IBCH) were established to account for background CO2 production, viability of the inoculum, and the toxicity of Rhodiantal Original IBCH, respectively. Test flasks were incubated aerobically in the dark for a period of 56 days.

The mean cumulative net percent CO2 evolved (percent biodegradation) from the aqueous test medium fortified with Rhodiantal Original IBCH at 10 mg C/L was -25.38% on day 56. The negative value indicates that the CO2 evolution was less than the inoculum controls. The toxicity control on day 14 was 44.99%, which indicates that Rhodiantal Original IBCH was not toxic (i.e., < 25%) to the inoculum in the test medium according to OECD Guideline 301B.

The cumulative net percent CO2 evolved from the procedural control was 66.12% of theoretical by day 14, thus meeting the “pass” criteria of the test (reaching 60% or greater CO2 evolution by day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

The difference between the replicates at the plateau and at the end of the study did not exceed 20% variation at any point during the study.

The control blanks did not exceed 40 mg evolved CO2 per liter.

Based on the extent of CO2 evolution during this study, Rhodiantal Original IBCH cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since it did not achieve 60% CO2 evolution within a 10-day window of reaching 10% biodegradation during the first 28 days of the study.

Description of key information

The test item was considered as not readily biodegradable (0% biodegradation (CO2 evolution) in 56 days).

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable

Additional information

A study, scored as Klimisch 1 and flagged as a key study, is available on Rhodiantal original IBCH giving 0% biodegradation in 56 days and revealing that Rhodiantal original IBCH is not readily biodegradable (Smithers Viscient, 2017).

Simulations test was initially planned in order to define if the substance is P and/or vP. However based on laboratories feedback, the study is considered to be not technically feasible and the OECD 307 testing proposal is withdrawn.

The substance is thus considered as vP (and P) without performing complementary test.

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