(2S)-3-Methyl-2-[N-({2'-(1H-1,2,3,4-tetrazol-5-yl)-[1,1-biphenyl]-4-yl}methyl)pentanamido]alkanoic acid

Adsorption / desorption

Link to relevant study record(s)

Reference

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Marc - July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.1110 (Activated Sludge Sorption Isotherm)

Qualifier:

according to guideline

Guideline:

OECD Guideline 106 (Adsorption - Desorption Using a Batch Equilibrium Method)

GLP compliance:

yes

Type of method:

HPLC estimation method

Media:

sewage sludge

Radiolabelling:

yes

Test temperature:

8°C

Details on study design: HPLC method:

Column
Stationary phase Symmetry shield RP-18
Dimensions 150 x 4.6 mm; dp = 5 um
Brand Merck, Darmstadt, Germany
Mobife phase 50/50/0.05 (vlvlv) acetonitrile/Milli-Q water/phosphoric acid
Flow 1 mL/min
Detection UV-detector (wavelength of 210 nm)
Radio-detector
Injection volume 100ul
Autosampler temperature 8°C

Analytical monitoring:

yes

Details on matrix:

Sludge preparation
Each of the sewage sludges (60 L) was sampled in two 30 L jerry-cans from three different wastewater treatment plants on March 31, 2006 and on April 3, 2006. The wastewater treatment plants included an aerobic plant treating domestic and industrial wastewater (Tilburg), an aerobic plant treating predominantly domestic wastewater (St Oedenrode) and a anaerobic plant treating exclusively (food) industrial wastewater ('s-Hertogenbosch).
After arrival at NOTOX, the sludges were allowed to settle at ambient temperature for 30 minutes. The water layers were separated as much as possible from the precipitated sludges and discarded. The sludges were-centrifuged (1700 g, 20°C, 5 min) and the-supernatant was decanted. The sludges were frozen at -80°C and subjected to freeze-drying. After freeze-drying, the sludges were sieved-over a 2 mm sieve.
In order to exclude the possibility of biodegradation of the test substance during the test, freezedried sludges were irradiated at Isotron Nederland B.V., Ede, The Netherlands. The materials were kept in plastic containers and were subjected to a dose of 25.0 kGy average in a JS6500 Tote Box Irradiator. Although irradiation was not performed under GLP- conditions, Isotron Nederland B.V. is certified with the national and international Quality Assurance System Standards NEN-ISO 9002, NEN4SO 14001 and EN 46002.
The sludges were stored in closed plastic containers at ambient temperature. Their moisture content was determined at NOTOX. % Organic matter (loss on ignition), pH and particle size distribution of the sludges were determined by National Resource Management Ltd (NRM), Berkshire, UK.
Stock and spike solutions
Stock solutions of 14C-labelled substance (VAA489 VAL) were prepared in acetonitrile. Spike solutions were prepared by diluting the stock solutions at least a hundred fold with a 0.01 M CaCl2 solution (resulting in an organic solvent content of less than 1% in spike solutions and less than 0.1% in test solutions).
Stock and spike solutions were prepared on the day of spiking. The radiochemical purity of the stock solution and spike solution was checked by HPLC.

Details on test conditions:

Determination of an appropriate sludge:solution ratio
A stock solution containing 3.35 MBq/mL 14C-labeled VAA489 VAL in acetonitrite was prepared. This is equivalent to 989 mg/L VAA489 VAL. A spike solution was prepared by diluting the stock solution with 0.01 M CaCl2 solution to 10.2 mg/L VAA489 VAL (34.6 kBq/mL based on triplicate anaiysis by LSC; RSD=2.4%). The pH of the spike solution was 6.2
Sludge:sofution ratio's of 1: 100 and 1:500 were investigated in order to determine an appropriate ratio for the adsorption kinetics and isotherms experiments. Approximately 40.5 ml of 0.01 M CaCl2 solution was added to 0.45 g sludge for a sludge:solution ratio of 1: 100 and 0.09 g sludge for a sludge:soiution ratio of 1 :500 in poiyethylene centrifuge-tubes. The sludges were equilibrated overnight on a roller mixer at 20 ± 1°C in the dark. After equilibration, the samples were spiked with approximately 4.5 g spike solution, such that the final VAA489 VAL concentration in the test solutions was approximately 1.0 mg/L For each sludge, a blank sample was included using a known amount of 0.01 M CaCl2 solution without test substance. A control sample without sludge was included to verify the initial concentration.
The samples were placed on a roller mixer at 20 ± 1°C in the dark. After 24 hours of contact time, the containers were removed from the roller mixer and centrifuged for 5 minutes at 756 g (the control was not centrifuged). The supernatants were removed and 100 uL aliquots were taken for LSC analysis.
Determination of equilibrium time (adsorption and desorption kinetics)
A stock solution containing 3.78 MBq/mL 14C-labelled VAA489 VAL in acetonitrile was prepared. This is equivalent to 1115 mg/L VAA489 VAL. A spike solution was prepared by diluting the stock solution with 0.01 M CaCl2 solution to 11.5 mg/L VAA489 VAL (38.9 kBq/mL based on triplicate analysis by LSC; RSD = 0.8%). The pH of the spike solution was 5.9.
Based on the experiment described, a sludge:solution ratio of 1:50 was selected for all sludges. Sludge:O.01 M CaCl2 slurries (approximately 0.9 g sludge and 40.5 ml 0.01 M CaCl2 solution in polyethylene centrifuge tubes) were equilibrated on a roller mixer at 20 ± 1°C in the dark for 3 days prior to spiking. The adsorption-desorption kinetics experiment was initiated by adding a known volume of approximately 4.5 ml- of spike solution to the preequilibrated sludge slurries. Hence, the initial concentration of VAA489 VAL in the solution was approximately 1.1 mg/L.
For each sludge, a blank sample was included using a known amount of approximately 45 mlof 0.01 M CaCl2 solution without test substance. Two control samples were also included, containing known amounts of approximately 40.5 ml- 0.01 M CaCl2 solution and 4.5 ml- spike solution but no sludge.
The samples were placed on a roller mixer at 20 ± 1°C in the dark. At the adsorption sampling times (3, 6, 24 and 48 hours), the sludge slurries were removed from the roller mixer and centrifuged for 5 minutes at 1700 g (the controls were not centrifuged). After centrifugation, a 100 ul- aliquot of the supernatant was taken from each sample including blanks) for determination of activity by LSC. Additionally, at sampling time 48 hours, 5 ml- subsamples were taken for HPLC-analysis. Directly after sampling, the sludge slurries were mixed well and placed back on the roller mixer until the next sampiing event. At the end of the adsorption phase, the remaining supernatant was decanted and weighed. Subsequently, an approximately equal weight of fresh 0.01 M CaCl2 solution was added to the sludge. The vials were closed and placed on the roller mixer. At the desorption sampling times (3, 6, 24 and 48 hours), the sludge slurries were removed from the roller mixer and centrifuged (5 minutes at 1700 g). After centrifugation a 100 uL aliquot of the supernatant was taken from each sample (including blanks) for the determination of activity by LSC. Additionally at sampling time 48 hours, 5 ml subsamples were taken for HPLC-analysis. After the final desorption sampling event, the remaining supernatant was decanted and weighed.
After the adsorption and desorption phase of the kinetics experiment, the pH of the supernatants (one replicate of each sludge) was determined on the day of decanting. The supernatants were stored in the deep freezer.
After removal of the supernatant at the end of the adsorption/desorption period, the sludge was allowed to dry to the air for 3-5 days (room temperature). Three subsamples of approximately 0.2 g were combusted.
The adsorption part was conducted in duplicate (replicate A and B) for each sludge. The desorption part was conducted with replicates B only. Mass balances were determined after the adsorption part (replicate A) and after the desorption part (replicate B) for each sludge. Mass balances included activity in samples taken for analysis, activity in decanted supernatant after adsorption and/or desorption and activity in sludge (determined by combustion).
Determination of adsorptionldesorption isotherms
A stock solution containing 8.11 MBq/mL 14C-labelled VAA489 VAL in acetonitrile was freshly prepared on the day of performance of the experiment. This is equivalent to 2394 mg/L 14C-abelled VAA489 VAL. Spike solutions were prepared by diluting the stock solution with 0.01 M CaCl2 solution to concentrations of 0.239 mg/L (0.810 kBq/mL; RSD=0.56%), 1.17 mg/L (3.95 kBq/mL; RSD=0.63%), 2.42 mg/L (8.18 kBq/mL; 12.3 mg/L (41.6 kBq/mL; RSD=0.91%) and 23.8 mg/L (80.7 kBq/mL; RSD=1.4%) based on triplicate analysis by LSC.
Sludge:0.01 M CaCl2 slurries (approximately 0.9 g sludge and 40.5 ml 0.01 M CaCl2 solution in polyethylene centrifuge tubes) were equilibrated at 20 ± 1°C in the dark on a roller mixer for 3 days, prior to spiking. The adsorption isotherms experiment was initiated by adding a known volume of approximately 4.5 ml of the different spike solutions to ten pre-equilibrated sludge slurries of each sludge (two replicates per concentration). In this way, initial VAA489 VAL concentrations of approximately 0.02, 0.12, 0.24, 1.2 and 2.4 mg/L were obtained. For each sludge a blank sample was included using a known amount of 0.01 M CaCl2 solution and no test substance.
The samples were incubated on a roller mixer at 20 ± 1°C in the dark. After 24 hours of contact time, the sludge:test solution slurries were removed from the roller mixer and centrifuged for 5 minutes at 1700 g (the controls were not centrifuged). The activity in 100 uL of supernatant was determined by LSC and the supernatants were decanted and weighed. The decanted supernatant was replaced by an approximately equal, known volume of fresh 0.01 M CaCl2 solution. The sludge slurries were mixed well and placed on a shaker at 20 ± 1°C for 24 hours. The sludge slurries were centrifuged (5 minutes at 1700 g). The activity in 100 ul of supernatant was determined by LSC.
The adsorption and desorption parts of the isotherms experiments were conducted in duplicate for each sludge. The pH of the supernatants after the adsorption and desorption parts of the experiment (one replicate of each sludge at the highest and lowest concentration level) was determined after decanting. The supernatants were stored in the deep freezer.

Key result

Sample No.:

#1

Type:

other: Freundlich equation

Value:

24 other: cm3/g

pH:

6

Temp.:

20 °C

Matrix:

sludge Tilburg

Key result

Sample No.:

#2

Type:

other: Freundlich equation

Value:

30 other: cm3/g

pH:

6

Temp.:

20 °C

Matrix:

sludge St Oedenrode

Key result

Sample No.:

#3

Type:

other: Freundlich equation

Value:

34 other: cm3/g

pH:

6

Temp.:

20 °C

Matrix:

Sludge s'-Hertogenbosch

Concentration of test substance at end of adsorption equilibration period:

The amount of VAA489 VAL adsorbed to sludge ranged from 21% to 49%.

Concentration of test substance at end of desorption equilibration period:

The amount of material desorbed from the sludge was in the range of 34 to 46%.

Transformation products:

not specified

Validity criteria fulfilled:

yes

Conclusions:

VAA489 VAL adsorption isotherms could be described by the Freundlich equation. KF om ads values were 24 cm3/g (Sludge [ - Tilburg), 30 cm3/g (Sludge Il - St Oedenrode) and 34 cm3/g (Sludge Ill - s'-Hertogenbosch). Based on these results, VAL489 VAA can be considered slightly mobile in sludge.

Executive summary:

VAA489 VAL adsorption isotherms could be described by the Freundlich equation. K_{F om} ^ads values were 24 cm3/g (Sludge [ - Tilburg), 30 cm3/g (Sludge Il - St Oedenrode) and 34 cm3/g (Sludge Ill - s'-Hertogenbosch). Based on these results, VAL489 VAA can be considered slightly mobile in sludge.

Description of key information

VAA489 VAL adsorption isotherms could be described by the Freundlich equation. KF om ads values were 24 cm3/g (Sludge [ - Tilburg), 30 cm3/g (Sludge Il - St Oedenrode) and 34 cm3/g (Sludge Ill - s'-Hertogenbosch). Based on these results, VAL489 VAA can be considered slightly mobile in sludge.

Key value for chemical safety assessment

Koc at 20 °C:

50