Reaction mass of 3,4,5-trichlorophthalic acid, strontium salt and 3,4,6-trichlorophthalic acid, strontium salt and 3,4-dichlorophthalic acid, strontium salt and 3,5-dichlorophthalic acid, strontium salt and 3,6-dichlorophthalic acid, strontium salt and 3-chlorophthalic acid, strontium salt and 4,5-dichlorophthalic acid, strontium salt and 4-chlorophthalic acid, strontium salt

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-24 to 2019-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Aerobic activated sludge (microorganisms from a domestic wastewater treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

102.9 mg/L

Based on:

ThOD

Remarks:

63.0 mg/L corresponding to an oxygen demand of about 102.9 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Conditions
Surrounding Type: Climatised chamber
Temperature: 22°C ± 1°C
Light Conditions: Darkness
pH-Value of Test Solutions: 7.6 (measured at the start of the test)
7.5 to 8.1 (measured at the end of the test)
Recording: Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and were not reported.)
Reconstituted Test Water
Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume

In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
The pH was adjusted to 7.6 with 1m HCl.

Preparation of Test Flasks
The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.

Incubation
The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

Validity Criteria of the Study
Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 10 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
pH-Value: The pH-value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.
The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation.
Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 0%.
The validity criterion was fulfilled.
Toxicity Control: If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 61% at day 14; the test item was not inhibitory.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Percentage Biodegradation:
The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.
The mean biodegradation percentage at the end of the 28-day exposure period was 0% (ThODNH4).

Conclusion:
The degradation rate of strontium dichlorophthalate (Strodip) never reached 60%.
Therefore, strontium dichlorophthalate (Strodip) is considered to be not readily biodegradable.

Results with reference substance:

Percentage Biodegradation:
The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 94% after 28 days of incubation.

Conclusion:
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Table 1. Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	0	0	0	0	45	0	40
2	0	0	0	0	80	0	65
3	0	0	5	0	115	0	85
4	0	0	5	5	125	0	105
5	5	0	5	5	130	0	115
6	5	5	5	5	135	0	120
7	5	5	5	5	135	0	125
8	5	5	10	5	140	0	130
9	5	5	10	10	145	0	135
10	5	5	10	10	145	0	140
11	5	10	10	10	145	0	150
12	5	10	10	10	145	0	150
13	5	10	10	10	145	0	150
14	5	10	10	10	145	0	155
15	5	10	10	10	145	0	155
16	5	10	10	10	150	0	155
17	5	10	10	10	150	0	155
18	5	10	10	10	150	0	160
19	5	10	10	10	150	0	160
20	5	10	10	10	150	0	160
21	5	10	10	10	150	0	165
22	10	10	10	10	150	0	165
23	10	10	10	10	155	0	165
24	10	10	10	10	155	0	165
25	10	10	10	10	160	0	165
26	10	10	10	10	165	0	165
27	10	10	10	10	165	0	165
28	10	10	10	10	170	0	165

Flasks 1 and 2: Strontium dichlorophthalate (Strodip);  

Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control);Flask 6: abiotic control

Flask 7: toxicity control

Table 2.Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time	Percentage Biodegradation
(Days)	Strontium dichlorophthalate (Strodip)^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	26	17
2	0	0	47	28
3	0	0	66	35
4	0	0	70	42
5	0	0	73	47
6	0	0	76	49
7	0	0	76	51
8	0	0	78	52
9	0	0	79	53
10	0	0	79	55
11	0	0	79	59
12	0	0	79	59
13	0	0	79	59
14	0	0	79	61
15	0	0	79	61
16	0	0	82	61
17	0	0	82	61
18	0	0	82	64
19	0	0	82	64
20	0	0	82	64
21	0	0	82	66
22	0	0	82	66
23	0	0	85	66
24	0	0	85	66
25	0	0	88	66
26	0	0	91	66
27	0	0	91	66
28	0	0	94	66

 ¹ThOD_NH4of Strontium dichlorophthalat (Strodip): 0.612 mg O₂/mg test item
 ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of strontium dichlorophthalate (Strodip) never reached 60%.
Therefore, strontium dichlorophthalate (Strodip) is considered to be not readily biodegradable.

Executive summary:

The biodegradation of strontium dichlorophthalate was studied over a period of 28 days in a suspension in mineral medium inoculated with activated sludge, in accordance with the OECD test guideline 301 F "Manometric Respirometry Test" and in complpiance with OECD-GLP standards. Aerobic activated sludge was supplied by a domestic wastewater treatment plant of Rossdorf, Germany. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as procedure control. The initial concentration of the test item in the medium was 102.9 mg/L corresponding to an oxygen demand of about 170.7 mg/L (ThOD_NH4). The test was conducted at 22 ° in darkness. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

The mean biodegradation at test end after 28 days was 0% (ThOD_NH4).

Therefore, strontium dichlorophthalate (Strodip) is considered to be not readily biodegradable based on ThOD_NH4.

The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 94% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 61% (ThOD_NH4) biodegradation was noted within 14 days and 66% (ThOD_NH4) biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days. 

Description of key information

In a Manometric Respirometry Test according to OECD guideline 301F the biodegradation of strontium dichlorophthalate was studied. The test substance is not readily biodegradable under the employed test conditions as no biodegradation was observed during the 28 days exposure time.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater