Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Adsorption / desorption

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-02 to 2013-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline with appropriate methods, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD 106 (2000) and Council Regulation (EC) No. 440/2008, C.18 (2001)

Deviations:

no

Remarks:

The study was based on these guidelines. Adaptations were necessary due to the test item specific physico-chemical properties.

GLP compliance:

yes

Type of method:

batch equilibrium method

Media:

soil

Radiolabelling:

no

Test temperature:

21 +- 2°C
Minimum: 19.0°C
Maximum: 21.3°C

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
10 mg/L for adsorption kinetics and desorption additional 50 and 100 mg/L for concentration dependency (adsorption isotherms) and 50 mg/L for
the impact of Ca2+; stock solutions of 1 g and 10 g test item /L were prepared in HPLC water.

- Sample preparation:
Equilibrated replicates were centrifugated (10 min. at 10000 g) prior to fortification. Prior to photometrical analysis replicates were centrifugated
(10 min. at 10000 g) and filtrated (0.45 µm).

- Sampling interval:

Replicates (adsorption kinetics):
Duplicates per sampling point for the adsorption kinetics. Duplicate samples for eurosoils 2 – 4 were agitated for 2, 4, 6, 24, 30 and 48h. In the case
of eurosoil 1 samples were agitated for 3, 5, 7, 24 and 30h. 0.4 mL of the stock solution (1 g/L) were added to the test systems consisting of 1 g soil
and 39.6 mL aqueous medium.

Replicates (desorption):
The replicates of the adsorption kinetics which had been agitated for 24h were used for this purpose. Therefore, the aqueous medium was decanted
into a graduated cylinder after centrifugation and the volume was determined. After that fresh aqueous medium was added to the test systems to a
final volume of 40 mL. The samples were agitated for 24h.

Replicates (adsorption isotherms):
Duplicate samples were agitated for 24h. Aliquots (0.2 and 0.4 mL) from the stock solution with a concentration of 10 g test item/L were used for
fortification. In addition, 0h samples were prepared as reference samples by spiking 0.05 mL stock solution and 0.05 mL HPLC water and 0.1 mL of
the stock solutions into 9.9 mL aqueous medium, respectively.


Replicates (Ca2+ dependency):
Duplicate samples were prepared by adding 0.2 mL of the stock solution with a concentration of 10 g test item/L into aqueous media with
concentrations of 40 and 400 mg CaCl2/L. These samples were agitated for 24h. 0h samples in these aqueous media were prepared by spiking 29.
85 mL with 0.15 mL of the stock solution with a concentration of 10 g/L.
No precipitation after addition of the test item to the equilibrated aqueous media, which contained the corresponding concentrations of CaCl2, was
observed. Furthermore no precipitated phase of the test item after agitation of these samples was observed during the study.


TEST ITEM CONTROLS (adsorption kinetics):
Furthermore, test item controls (duplicates) were prepared to investigate the test item stability during agitation on the overhead shaker. Therefore,
the supernatant was removed and fortified to a concentration of 10 mg test item/L by spiking 29.7mL with 0.3 mL of the stock solution with a
concentration of 1 g/L. An aliquot of these samples was used for the determination of the reference value (0h value) via photometry. The remaining
part of the samples was agitated for 48h or 30h in the case of eurosoil 1.

BLANK SAMPLES:
Blank samples for the adsorption kinetics and the investigations about concentration dependency were prepared as described for the test item
replicates but without fortification with the test item. Blank samples for experiments about Ca2+ dependency were obtained from the 0h samples
prior to fortification with the test item. Blank samples for desorption experiments were obtained from a blank sample of the adsorption kinetic
experiments which had been treated and agitated analogously to the corresponding test item replicates. Aliquots of these samples were analysed
for background correction during photometric analysis.


- Sample storage before analysis: All samples were stored at room temperature until analysis.

Details on matrix:

Standard eurosoils no. 1, 2, 3, 4 and 5 in contact to demineralised water were used. These soils have varying adsorption capacities in relation to their content of organic matter, clay and metal oxides as well as pH and cation exchange capacity. All soils were received air dried and sieved to a
maximum particle size of 2 mm.
The physico-chemical characteristics of the different soils are given in the Table below.

Eurosoils
1 2 3 4 5
FAO soil unit 1) Vertic Rendzina Dystric Orthic Orthic
Cambisol Dystric Cambisol Luvizol Podzol

pH 1) 6.2 8.1 6.2 7.5 4.1
Total Carbon [%] 1) 3.39 10.81 3.25 1.45 6.43
Organic Carbon [%] 1) 3.27 3.72 3.01 1.31 5.96
Clay (<0.002 mm) [%] 2) 74.6 29.0 17.3 20.9 4.3
Silt (0.002-0.063 mm) [%] 2) 24.5 56.1 32.8 73.2 8.8
Sand (0.063-2 mm) [%] 2) 1.0 15.0 50.0 6.0 87.0

1) Gawlik and Muntau, Eurosoils II Laboratory and Reference Materials for Soil-related Studies, Environment Institute 1999
2) determined at INSTITUT KOLDINGEN AGROLAB LABORGRUPPE (non-GLP), mean value of two replicates related to Dry weight


Reason for the selection:
These soils varied considerably in their sorption relevant physico-chemical properties. Therefore, these soils were suitable for the conduction of the study because all soil parameters with impact on the adsorption / desorption behaviour of a chemical substance were considered.

Origin of soils:
European Commission, Joint Research Centre, INSTITUTE FOR REFERENCE MATERIALS AND MEASUREMENTS IRMM, Retieseweg, B-2440 Geel, Belgium

Storage at test facility:
Room temperature, dark, in closed containers (brown glass bottles).

Expiry date:
Eurosoil 1, 2, 3 and 4: 2013-08-21
Eurosoil 5: 2013-09-30

Details on test conditions:

TEST CONDITIONS
- pH: The pH values of the aqueous media of the test systems were measured before and after equilibration with the corresponding soils as well as
after addition of the test item. The pH values ranged between 4.15 and 8.29 after equilibration with the soils and from 4.20 to 8.10 after addition of
the test item. Among the soils used for the experiment, eurosoil 5 was the most acidic soil, while the most basic soil was eurosoil 2. The test item,
HEBMP Cyclic Form, pH-neutral, did not have a significant impact on the pH value of the test system.

pH values of the aqueous Media:
(Values before and after soil contact as well as after addition of the test item are given. Mean values of two replicates are described.)


Eurosoils
1 2 3 4 5
before soil contact 6.25 6.18 6.62 6.86 6.13
after soil contact 6.16 8.29 6.38 7.46 4.15
after addition of the test item 6.28 8.10 6.39 7.63 4.20


TEST SYSTEM
- Type, size and further details on reaction vessel: 50 mL centrifugation tubes, VWR
- Soil/solution ratio: 1 g soil and 40 mL solution
- Aqueous media: Tap water demineralised by reverse osmosis, additional 40 and 400 mg/L CaCl2 for investigation of impact of Ca2+ on adsorption
- Equilibration: The test systems were equilibrated with aqueous medium by agitation overnight (minimum 12h).
- Measuring equipment: Photometer Nanocolor UV/VIS, Machery & Nagel
- Method of preparation of test solution: Prior to photometrical analysis replicates were centrifugated (10 min. at 10000 g) and filtrated (0.45 µm).
- Are the residues from the adsorption phase used for desorption: yes

Computational methods:

Determination of adsorption kinetics, % of adsorption, adsorption isotherms and % of desorption was performed according to the equations given
below.
These endpoints were determined by the indirect method. Only the test item concentrations in the aqueous media were monitored. Decline of the test item in the aqueous media by other reasons than adsorption on soil could be ruled out as demonstrated by preliminary experiments
(Tier 1, non-GLP). Results of test item controls during these experiments with eurosoils 1 and 5 showed that adsorption on test vessels did not occur.
In addition, test item controls were prepared for all soils and treated as the test item replicates during experiments for determination of adsorption
kinetics in the definitive study.


% Adsorption: [%] Adsorption = (m0 - madsaq(ti)) / m0 * 100

m0 = initial nominal mass of test item in aqueous phase [mg]
madsaq(ti) = mass of test item measured in the aqueous phase at the point of time ti [mg]


Distribution Coefficient KD (Adsorption):

KD = (madss (eq)/madsaq) * (V aq / m soil) [mL/g]

madss = mass of test item in the soil phase at equilibrium [mg]
madsaq = mass of test item in the aqueous phase at equilibrium [mg]
m soil = used amount of soil [g]
V aq = used volume of aqueous phase [mL]


Adsorption Isotherms:
The adsorbed mass per unit mass of soil was plotted as a function of the calculated equilibrium concentration of the test item. The FREUNDLICH
adsorption isotherm equation relates the amount of the test item adsorbed to the concentration of the test item in solution at equilibrium.

Cadss (eq) = KadsF * Cadsaq (eq)^(1/n)

Cadss (eq) = concentration of the test item adsorbed on the soil at adsorption equilibrium [mg/g]
KadsF = Freundlich adsorption coefficient mg^(1-1/n)(mL)^(1/n) g^-1]
Cadsaq (eq) = concentration of the test item in the aqueous phase at adsorption equilibrium [mg/mL]
1/n = regression constant

In its linear form the FREUNDLICH adsorption equation is as follows:

log Cadss (eq) = log KadsF + (1/n) * log Cadsaq (eq)

The reciprocal regression coefficient was directly deduced from the slope of the linear regression if sufficient linearity was obtained.



% Desorption:
[%] Desorption = (mdesaq / (m0 - madsaq)) * 100%

m0 = initial nominal mass of test item [mg]
madsaq = mass of test item measured in the aqueous phase after the adsorption step [mg]
mdesaq = mass of test item measured in the aqueous phase after the desorption step [mg]


Software:
The data for the tables in the report were computer generated (EXCEL, MICROSOFT CORPORATION) and rounded for presentation from the full
derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Type:

Kd

Value:

87 L/kg

Temp.:

20 °C

% Org. carbon:

3.27

Remarks on result:

other: Eurosoil 1; Temperature: 19.0 - 21.3°C

Type:

Kd

Value:

14 L/kg

% Org. carbon:

3.72

Remarks on result:

other: Eurosoil 2; Temperature: 19.0 - 21.3°C

Type:

Kd

Value:

12 L/kg

% Org. carbon:

3.01

Remarks on result:

other: Eurosoil 3; Temperature: 19.0 - 21.3°C

Type:

Kd

Value:

5 L/kg

% Org. carbon:

1.31

Remarks on result:

other: Eurosoil 4; Temperature: 19.0 - 21.3°C

Type:

Kd

Value:

3 L/kg

% Org. carbon:

5.96

Remarks on result:

other: Eurosoil 5; Temperature: 19.0 - 21.3°C

Type:

Kd

Value:

>= 90 - <= 94 L/kg

% Org. carbon:

3.27

Remarks on result:

other: Eurosoil 1; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 8 - <= 9 L/kg

% Org. carbon:

3.72

Remarks on result:

other: Eurosoil 2; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

18 L/kg

% Org. carbon:

3.01

Remarks on result:

other: Eurosoil 3; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 6 - <= 7 L/kg

% Org. carbon:

1.31

Remarks on result:

other: Eurosoil 4; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 26 - <= 27 L/kg

% Org. carbon:

5.96

Remarks on result:

other: Eurosoil 5; CaCl2 present in aqueous phase at 400 mg/L

Recovery of test material:

not applicable. HEBMP could not be analysed in the solid phase due to effectively irreversible
binding.

Transformation products:

not measured

Details on results (Batch equilibrium method):

-Eurosoil 1 :
Adsorption kinetics:
% adsorption over the treatment of 30h were 70 and 67% for the two replicates after 30h of agitation. Adsorption increased up
to 47% after 3h of agitation and up to 67% after 24h of agitation. The further increase in adsorption between 24 and 30h of agitation was not
significant. Based on the mean value for the adsorbed fractions between 24 and 30h a distribution coefficient KD of 87 mL/g was calculated.
Adsorption isotherms:
The adsorbed amount of the test item on soil increased with higher loading level of the test item in the aqueous medium. Nevertheless, the
adsorption behaviour was not linear and the distribution coefficients KD after 24h decreased from a maximum of 85 mL/g at 10 mg test item/L to
16 mL/g at a concentration of 100 mg test item/L. The graphical expression of the adsorption behaviour was characterised by a coefficient of
determination R2 of 0.9939. The reciprocal regression coefficient 1/n was 0.4636 indicating that adsorption behaviour was not linear in the
investigated concentration range.
Impact of Ca2+ on adsorption:
The results demonstrate the increase of the distribution coefficient KD with increasing Ca2+ concentrations. Distribution coefficients KD from
maximum 25 mL/g in demineralised water steadily increased to a maximum value for KD of 94 mL/g when a 400 mg/L solution of CaCl2 as
aqueous medium was used for the adsorption experiment.
Desorption:
Up to 23% of the adsorbed fractions were desorbed over a period of 24h.

- Eurosoil 2
Adsorption kinetics: % of adsorption over the treatment of 48h were between 17 and 19% after 24 and 30h of agitation indicating equilibrium. But
with further agitation the adsorption again increased and values of 24 and 26% for the two replicates were obtained after 48h. This increase was
clearly visible by a second slope in the graph of the adsorption kinetics. Based on the mean value for the adsorbed fraction (25%) after 48h a
distribution coefficient KD of 14 mL/g was calculated though the system was not in equilibrium. The mean recovery of 100% for the test item
controls indicated that test item adsorption on the test vessels did not occur and only adsorption on soil was responsible for the decline of the
test item in the aqueous phase.
Adsorption isotherms: The distribution coefficients KD decreased from a maximum of 9 mL/g at 10 mg test item/L to 1 mL/g at a concentration
of 100 mg test item/L indicating no linear adsorption behaviour in the investigated concentration range. No linearity was obtained in the
graphical expression of the concentration dependency. Therefore, no regression coefficient 1/n was calculated.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item on this soil but
the impact was first visible with the highest loading level of 400 mg/L CaCl2. An increase of the distribution coefficient KD from maximum
4 mL/g in demineralised water to a maximum value for KD of 9 mL/g for the treatment with 400 mg/L CaCl2 was determined.
Desorption: Up to 60% of the adsorbed fractions were desorbed over a period of 24h.

-Eurosoil 3:
Adsorption kinetics: % of adsorption over the treatment of 48 h were between 21 and 26% in equilibrium. The test system was in equilibrium after
30h of agitation. Based on the mean value for the adsorbed fractions, taking into account the values obtained after 30 and 48h of agitation, a
distribution coefficient KD of 12 mL/g was calculated. The mean recovery of 97% for the test item controls indicated that test item adsorption on
the test vessels did not occur and only adsorption on soil was responsible for the decline of the test item in the aqueous phase.
Adsorption isotherms: The distribution coefficients KD decreased from a maximum of 10 mL/g at 10 mg test item/L to 4 mL/g at a
concentration of 100 mg test item/L indicating no linear adsorption behaviour in the investigated concentration range. No linearity was obtained
in the graphical expression of the concentration dependency. Therefore, no regression coefficient 1/n was calculated.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ had an impact on the adsorption behaviour of the test item in this soil. Distribution
coefficients KD from maximum 3 mL/g in demineralised water steadily increased to a maximum value for KD of 18 mL/g when a 400 mg/L
solution of CaCl2 as aqueous medium was used for the adsorption experiment.
Desorption: Up to 48% of the adsorbed fractions were desorbed over a period of 24h.

-Eurosoil 4:
Adsorption kinetics: Up to 4% adsorption were determined over the treatment of 48h after 24h of agitation indicating equilibrium. But with further
agitation the adsorption again increased and values of 10 and 12% for the two replicates were obtained after 48h. This increase was clearly visible
by a second slope in the graph of the adsorption kinetics. Based on the mean value for the adsorbed fraction (11%) after 48h a distribution
coefficient KD of 5 mL/g was calculated though the system was not in equilibrium. The mean recovery of 98% for the test item controls indicated
that test item adsorption on the test vessels did not occur and only adsorption on soil was responsible for the decline of the test item in the
aqueous phase.
Adsorption isotherms: Independent on the concentration level the distribution coefficients KD were between of 1 and 2 mL/g. A steady increase in
the adsorbed mass on soil was observed. The increase in the adsorbed mass correlated approximately proportionally with the increase in the
loading level of the test item in the aqueous medium indicating slight linear adsorption behaviour. The graphical expression of the adsorption
behaviour was characterised by a coefficient of determination R2 of 0.9499 and the reciprocal regression coefficient 1/n was 1.2636.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item in this soil. An
increase of the distribution coefficient KD from maximum 2 mL/g in demineralised water to a maximum value for KD of 7 mL/g for the treatment with
400 mg/L CaCl2 was observed.
Desorption: Adsorption of the test item in this soil was of less relevance after 24h of agitation. The results for desorption were implausible because
more test item was desorbed than adsorbed over 24h. Therefore % of desorption was not calculated.

-Eurosoil 5:
Adsorption kinetics: Over the treatment of 48h no clear tendency for the adsorption behaviour of the test item in this soil was observed. The
maximum of mean adsorption was observed after 24h of agitation with 5 and 10%. Based on this mean value a distribution coefficient KD of 3 mL/g
was calculated.
Adsorption Isotherms: Independent on the concentration level the distribution coefficients KD were between 2 and 4 mL/g. A steady increase in the
adsorbed mass on soil was observed with higher test item loading level in the aqueous medium. Nevertheless, the adsorption behaviour was not
linear. No linearity was obtained in the graphical expression of the concentration dependency. Therefore, no regression coefficient 1/n was
calculated.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ had an impact on the adsorption behaviour of the test item in this soil. Distribution
coefficients KD from maximum 3 mL/g in demineralised water steadily increased to a maximum value for KD of 27 mL/g when a 400 mg/L solution
of CaCl2 as aqueous medium was used for the adsorption experiments.
Desorption: Adsorption of the test item in this soil was of less relevance after 24h of agitation. The results for desorption were implausible because
more test item was desorbed than adsorbed over 24h. Therefore % of desorption was not calculated.

Tables:

Eurosoil 1 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq[mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] maq [mg] msoil [mg] Adsorption [%]
1 0 2.00 0.400 0.000 0
2 0 2.02 0.400 0.000 0
1 3 1.07 0.213 0.187 47
2 3 1.09 0.217 0.183 46
1 5 0.94 0.187 0.213 53
2 5 0.96 0.191 0.209 52
1 7 0.86 0.171 0.229 57
2 7 0.91 0.181 0.219 55
1 24 0.67 0.133 0.267 67
2 24 0.69 0.137 0.263 66
1 30 0.61 0.121 0.279 70
2 30 0.66 0.131 0.269 67


Eurosoil 1 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium
and soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
10 0 1 2.00 0.400 0.000 - - - -
0 2 2.02 0.400 0.000 - - - -
24 1 0.67 0.133 0.267 -2.477 -0.549 67 85
24 2 0.69 0.137 0.263 -2.464 -0.556 66 81
50 0 1 9.89 2.000 0.000 - - - -
0 2 9.92 2.000 0.000 - - - -
24 1 6.23 1.258 0.742 -1.502 -0.105 37 25
24 2 6.52 1.317 0.683 -1.483 -0.141 34 22
100 0 1 19.99 4.000 0.000 - - - -
0 2 20.29 4.000 0.000 - - - -
24 1 14.39 2.858 1.142 -1.146 0.082 29 17
24 2 14.64 2.908 1.092 -1.139 0.063 27 16


Eurosoil 1 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 9.89 2.000 0.000 - -
Ca2+ 2 0 9.92 2.000 0.000 - -
1 24 6.23 1.258 0.742 37 25
2 24 6.52 1.317 0.683 34 22
40 mg/L
CaCl2
1 0 10.80 2.000 0.000 - -
2 0 10.73 2.000 0.000 - -
1 24 5.14 0.955 1.045 52 46
2 24 5.14 0.955 1.045 52 46
400 mg/L
CaCl2
1 0 10.60 2.000 0.000 - -
2 0 10.63 2.000 0.000 - -
1 24 3.29 0.620 1.380 69 94
2 24 3.39 0.639 1.361 68 90


Eurosoil 1 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 2.00 0.400 0.000 - -
2 0 2.02 0.400 0.000 - -
1 24h Adsorption 0.67 0.133 0.267 67 -
2 24h Adsorption 0.69 0.137 0.263 66 -
1 24h Desorption 0.29 0.058 0.209 - 22
2 24h Desorption 0.30 0.060 0.203 - 23


Eurosoil 2 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 2.10 0.400 0.000 0
2 0 2.13 0.400 0.000 0
1 2 1.94 0.367 0.033 8
2 2 1.92 0.363 0.037 9
1 4 1.87 0.354 0.046 12
2 4 1.90 0.359 0.041 10
1 6 1.87 0.354 0.046 12
2 6 1.86 0.352 0.048 12
1 24 1.76 0.333 0.067 17
2 24 1.73 0.327 0.073 18
1 30 1.74 0.329 0.071 18
2 30 1.71 0.323 0.077 19
1 48 1.57 0.297 0.103 26
2 48 1.60 0.303 0.097 24


Eurosoil 2 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
10 0 1 2.10 0.400 0.000 - - - -
0 2 2.13 0.400 0.000 - - - -
24 1 1.76 0.333 0.067 -2.080 -1.161 17 8
24 2 1.73 0.327 0.073 -2.087 -1.125 18 9
50 0 1 10.04 2.000 0.000 - - - -
0 2 9.54 2.000 0.000 - - - -
24 1 8.97 1.832 0.168 -1.339 -0.764 8 4
24 2 9.09 1.857 0.143 -1.333 -0.832 7 3
100 0 1 19.05 4.000 0.000 - - - -
0 2 18.84 4.000 0.000 - - - -
24 1 18.69 3.946 0.054 -1.006 -1.257 1 1
24 2 18.69 3.946 0.054 -1.006 -1.257 1 1


Eurosoil 2 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 10.04 2.000 0.000 - -
Ca2+ 2 0 9.54 2.000 0.000 - -
1 24 8.97 1.832 0.168 8 4
2 24 9.09 1.857 0.143 7 3
40 mg/L
CaCl2
1 0 10.40 2.000 0.000 - -
2 0 10.50 2.000 0.000 - -
1 24 9.42 1.803 0.197 10 4
2 24 9.42 1.803 0.197 10 4
400 mg/L
CaCl2
1 0 10.47 2.000 0.000 - -
2 0 10.37 2.000 0.000 - -
1 24 8.69 1.668 0.332 17 8
2 24 8.63 1.656 0.344 17 9


Eurosoil 2 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 2.05 0.400 0.000 - -
2 0 2.06 0.400 0.000 - -
1 24h Adsorption 1.73 0.337 0.063 16 -
2 24h Adsorption 1.74 0.339 0.061 15 -
1 24h Desorption 0.13 0.025 0.038 - 40
2 24h Desorption 0.19 0.037 0.024 - 60


Eurosoil 3 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 1.97 0.400 0.000 0
2 0 1.92 0.400 0.000 0
1 2 1.70 0.350 0.050 13
2 2 1.71 0.352 0.048 12
1 4 1.66 0.341 0.059 15
2 4 1.66 0.341 0.059 15
1 6 1.73 0.356 0.044 11
2 6 1.68 0.346 0.054 14
1 24 1.58 0.325 0.075 19
2 24 1.57 0.323 0.077 19
1 30 1.44 0.296 0.104 26
2 30 1.54 0.317 0.083 21
1 48 1.50 0.308 0.092 23
2 48 1.49 0.306 0.094 23


Eurosoil 3 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
10 0 1 1.97 0.400 0.000 - - - -
0 2 1.92 0.400 0.000 - - - -
24 1 1.58 0.325 0.075 -2.090 -1.116 19 9
24 2 1.57 0.323 0.077 -2.093 -1.104 19 10
50 0 1 9.42 2.000 0.000 - - - -
0 2 9.42 2.000 0.000 - - - -
24 1 8.78 1.864 0.136 -1.332 -0.858 7 3
24 2 8.74 1.856 0.144 -1.334 -0.832 7 3
100 0 1 19.90 4.000 0.000 - - - -
0 2 19.47 4.000 0.000 - - - -
24 1 18.02 3.662 0.338 -1.038 -0.462 8 4
24 2 17.84 3.625 0.375 -1.043 -0.417 9 4

Eurosoil 3 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 9.42 2.000 0.000 - -
Ca2+ 2 0 9.42 2.000 0.000 - -
1 24 8.78 1.864 0.136 7 3
2 24 8.74 1.856 0.144 7 3
40 mg/L
CaCl2
1 0 10.44 2.000 0.000 - -
2 0 10.47 2.000 0.000 - -
1 24 8.63 1.651 0.349 17 9
2 24 8.82 1.687 0.313 16 8
400 mg/L
CaCl2
1 0 10.63 2.000 0.000 - -
2 0 10.44 2.000 0.000 - -
1 24 7.37 1.399 0.601 30 18
2 24 7.31 1.388 0.612 31 18



Eurosoil 3 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 1.97 0.400 0.000 - -
2 0 1.92 0.400 0.000 - -
1 24h Adsorption 1.58 0.325 0.075 19 -
2 24h Adsorption 1.57 0.323 0.077 19 -
1 24h Desorption 0.13 0.027 0.048 - 36
2 24h Desorption 0.18 0.037 0.040 - 48


Eurosoil 4 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 2.06 0.400 0.000 0
2 0 1.95 0.400 0.000 0
1 2 2.00 0.399 0.001 0
2 2 2.02 0.403 -0.003 -1
1 4 1.97 0.393 0.007 2
2 4 1.97 0.393 0.007 2
1 6 1.95 0.389 0.011 3
2 6 2.00 0.399 0.001 0
1 24 1.97 0.393 0.007 2
2 24 1.92 0.383 0.017 4
1 30 1.87 0.373 0.027 7
2 30 1.90 0.379 0.021 5
1 48 1.81 0.361 0.039 10
2 48 1.76 0.351 0.049 12


Eurosoil 4 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
10 0 1 2.06 0.400 0.000 - - - -
0 2 1.95 0.400 0.000 - - - -
24 1 1.97 0.393 0.007 -2.008 -2.1441 2 1
24 2 1.92 0.383 0.017 -2.019 -1.7587 4 2
50 0 1 9.81 2.000 0.000 - - - -
0 2 9.72 2.000 0.000 - - - -
24 1 9.33 1.911 0.089 -1.321 -1.038 4 2
24 2 9.39 1.923 0.077 -1.318 -1.103 4 2
100 0 1 19.81 4.000 0.000 - - - -
0 2 19.66 4.000 0.000 - - - -
24 1 18.87 3.825 0.175 -1.019 -0.744 4 2
24 2 18.66 3.782 0.218 -1.024 -0.650 5 2

Eurosoil 4 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 9.81 2.000 0.000 - -
Ca2+ 2 0 9.72 2.000 0.000 - -
1 24 9.33 1.911 0.089 4 2
2 24 9.39 1.923 0.077 4 2
40 mg/L
CaCl2
1 0 10.67 2.000 0.000 - -
2 0 10.50 2.000 0.000 - -
1 24 9.71 1.835 0.165 8 4
2 24 9.84 1.859 0.141 7 3
400 mg/L
CaCl2
1 0 10.50 2.000 0.000 - -
2 0 10.44 2.000 0.000 - -
1 24 9.18 1.754 0.246 12 6
2 24 9.02 1.723 0.277 14 7


Eurosoil 4 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24 h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 2.06 0.400 0.000 -
2 0 1.95 0.400 0.000 -
1 24h Adsorption 1.97 0.393 0.007 2
2 24h Adsorption 1.92 0.383 0.017 4
1 24h Desorption 0.13 0.026 0.000 - n.c.
2 24h Desorption 0.14 0.028 0.000 - n.c.
n. c. = not calculated


Eurosoil 5 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 1.84 0.400 0.000 0
2 0 1.84 0.400 0.000 0
1 2 1.87 0.407 -0.007 -2
2 2 1.90 0.413 -0.013 -3
1 4 1.73 0.376 0.024 6
2 4 1.71 0.372 0.028 7
1 6 1.79 0.389 0.011 3
2 6 1.79 0.389 0.011 3
1 24 1.74 0.378 0.022 5
2 24 1.66 0.361 0.039 10
1 30 1.84 0.400 0.000 0
2 30 1.81 0.393 0.007 2
1 48 1.78 0.387 0.013 3
2 48 1.78 0.387 0.013 3


Eurosoil 5 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
10 0 1 1.84 0.400 0.000 - - - -
0 2 1.84 0.400 0.000 - - - -
24 1 1.74 0.378 0.022 -2.024 -1.656 5 2
24 2 1.66 0.361 0.039 -2.045 -1.400 10 4
50 0 1 9.71 2.000 0.000 - - - -
0 2 9.75 2.000 0.000 - - - -
24 1 9.00 1.850 0.150 -1.335 -0.817 8 3
24 2 8.97 1.844 0.156 -1.336 -0.799 8 3
100 0 1 19.75 4.000 0.000 - - - -
0 2 19.81 4.000 0.000 - - - -
24 1 18.72 3.786 0.214 -1.024 -0.662 5 2
24 2 18.69 3.780 0.220 -1.025 -0.650 6 2


For further tables see "Any other information on results inc. tables".

Eurosoil 5 – Impact of Ca²⁺on Adsorption

Values were calculated after 24h of agitation.

Replicate t [h] cmeasured [mg P/L] maq[mg] msoil [mg] Adsorption [%] KD [mL/g] without exogenous Ca^2+ 1 0 9.71 2.000 0.000 - - 2 0 9.75 2.000 0.000 - - 1 24 9.00 1.850 0.150 8 3 2 24 8.97 1.844 0.156 8 3 40 mg/L CaCl2   1 0 10.50 2.000 0.000 - - 2 0 10.60 2.000 0.000 - - 1 24 7.57 1.435 0.565 28 16 2 24 7.97 1.511 0.489 24 13 400 mg/L CaCl2   1 0 10.34 2.000 0.000 - - 2 0 10.60 2.000 0.000 - - 1 24 6.32 1.207 0.793 40 27 2 24 6.42 1.226 0.774 39 26

 

Eurosoil 5 – Desorption

Determination of the desorbed fractions after 24h adsorption and 24 h desorption.

 

Replicate	t [h]	cmeasured [mg P/L]	maq [mg]	m soil [mg]	Adsorption [%]	Desorption [%]
1	0	1.84	0.400	0.000	-
2	0	1.84	0.400	0.000	-
1	24h Adsorption	1.74	0.378	0.022	1
2	24h Adsorption	1.66	0.361	0.039	2
1	24h Desorption	0.40	0.087	0.000	-	n. c.
2	24h Desorption	0.35	0.076	0.000	-	n. c.

n. c. = not calculated

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The conclusions drawn from all experiments are as follows:

1) Distinct adsorption (> 20%) can only be expected in soils with a high clay content (> 29% clay amount of eurosoil 2). Based on the obtained data one cannot
deduce if silt or the sand fraction has a higher priority for the adsorption of the test item without exogenous Ca2+.

2) No linear adsorption behaviour was observed in the investigated concentration range (10 – 100 mg/L) for HEBMP Cyclic Form, pH-neutral in eurosoil 1 – 5.
A steady increase in the adsorbed amount was observed in all eurosoils except of eurosoil 2 when the test item loading level in the aqueous medium was
increased.

3) Ca2+ in the aqueous medium of the adsorption experiments enhances the adsorption behaviour of HEBMP Cyclic Form, pH-neutral. At least with the
highest loading level of 400 mg/L CaCl2 an increase for the distribution coefficients KD was observed in all tested soils. The presence of Ca2+ had a higher
impact on the increase of the distribution coefficients KD in eurosoils 3 and 5 than in eurosoils 2 and 4. These data let assume that sand is more preferred
as fraction for adsorption than the silt fraction in the presence of exogenous Ca2+.

4) The adsorption is partially reversible in eurosoils 1 - 3. No plausible values for desorption could be determined in eurosoils 4 and 5 because the degree of
adsorption was too low for an accurate determination of the % of desorption.

Executive summary:

The adsorption / desorption behaviour of HEBMP Cyclic Form, pH-neutral (batch no.SK233) in 5 different soils was determined following OECD guideline 106 and EC C.18.The study was conducted from 2012-11-02 to 2013-03-05, at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

The test item is an alkylphosphono oxazaphosphorin derivative.

Adsorption Kinetic in Eurosoils:

The table below shows the results obtained for the determination of distribution coefficients K_D in eurosoils 1 to 5. Adsorption experiments lasted 48h for eurosoils 2 – 5 and 30h for eurosoil 1. The highest K_D with 87 mL/g was obtained in eurosoil 1. This soil is characterised by a high clay content of 74.6%. Silt and sand are minor constituents of this soil at 24.5 and 1.0% w/w respectively. The distribution coefficients K_D in all others soils ranged between 3 and 14 mL/g. No correlation between the distribution coefficients K_D and the soil characteristics was visible.

Distribution CoefficientsK_D for HEBMP CyclicForm, pH-neutral in Eurosoils:

Adsorption experiments lasted 48h and 30h for eurosoil 1.

Application concentration 10 mg HEBMP Cyclic Form, pH-neutral/L

	Eurosoils
	1	2	3	4	5
KD [mL/g]	87	14	12	5	3

Adsorption Isotherms in Eurosoils:

The table below shows the results for the concentration dependency of adsorption. Therefore, additional concentration levels of 50 and 100 mg test item/L were tested, the degree of adsorption as mass adsorbed on soil was determined after 24h of agitation and compared with the values obtained in the lowest application concentration with 10 mg test item/L. A steady increase in the amount adsorbed on soil was observed when the loading level of the test item in the aqueous medium was enhanced except of eurosoil 2. In the latter one, the adsorbed amount increased first with a loading level of 50 mg test item/L but decreased below the level obtained with 10 mg test item/L when the concentration was doubled to 100 mg test item/L. Linearity in the graphical expressions was only obtained for eurosoil 1 and 4 and therefore regression coefficients1/n are given. Nevertheless, the adsorption behaviour is not linear in all soils.

Adsorbed Mass on Soils in Dependence on Test Item Concentration

Samples were agitated for 24h.

Total amount of test item applied per replicate in the different concentration levels:

0.4, 2, 4 [mg]

Test Item Concentration [mg/L]	Replicate	Eurosoils
		1	2	3	4	5
		msoil[mg]
10	1	0.267	0.067	0.075	0.007	0.022
	2	0.263	0.073	0.077	0.017	0.039
50	1	0.742	0.168	0.136	0.089	0.150
	2	0.683	0.143	0.144	0.077	0.156
100	1	1.142	0.054	0.338	0.175	0.214
	2	1.092	0.054	0.375	0.218	0.220
R^2		0.9939	n.d.	n.d.	0.9499	n.d.
Linearity		yes	no	no	yes	no
1/n		0.4636	n.d.	n.d.	1.2636	n.d

n.d.= could not be determined because no linearity was observed in the corresponding graphs

Eurosoils – Impact of Ca²⁺on Adsorption:

The table below summarises the values for the distribution coefficients K_D of HEBMP Cyclic Form, pH-neutral in the different eurosoils in dependence on increasing concentration of exogenous Ca²⁺ in the aqueous medium. Therefore, further adsorption experiments were performed with concentrations of 40 and 400 mg/L CaCl₂ in the aqueous medium and the resulting distribution coefficients K_D were compared with those obtained without exogenous Ca²⁺. The samples were agitated for 24h. The data show that exogenous Ca²⁺enhances the adsorption of the test item on soil. An exogenous level of 40 mg/L CaCl₂ was sufficient to enhance the adsorption of the test item in eurosoils 1, 3, 4 and 5. With further increase of the exogenous Ca²⁺ concentration to 400 mg/L CaCl₂, increased adsorption of the test item was also observed in eurosoil 2. Furthermore, the data indicate that the increase in the distribution coefficients K_D was higher in eurosoils 3 and 5 than in eurosoil 2 and 4. The latter ones have higher amounts of silt but lower amounts of sand. Therefore, one can conclude that the sand fraction is more preferred for adsorption of the test item in the presence of exogenous Ca²⁺.

Distribution Coefficients K_D for HEBMP Cyclic Form, pH-neutral in Eursoils in Dependence on exogenous Ca^{2 +}Concentrations:

Adsorption experiments lasted 24h.

Application concentration 50 mg HEBMP Cyclic Form, pH-neutral/L

Concentration exogenous CaCl2 [mg/L]	Replicate	Eurosoils
		1	2	3	4	5
		KD [mL/g]
0	1	25	4	3	2	3
	2	22	3	3	2	3
40	1	46	4	9	4	16
	2	46	4	8	3	13
400	1	94	8	18	6	27
	2	90	9	18	7	26

Eurosoils – Desorption:

The table below shows the results of the desorption experiments. Desorption experiments were performed after an adsorption period of 24h and lasted 24h. The data indicate that adsorption is at least partially reversible in eurosoil 1 – 3 with % of desorption between 22 and 60%. The results for eurosoils 4 and 5 are not plausible because the desorbed amount was higher than the adsorbed amount. One has to consider that the experimental error for the determination of % desorption becomes largest in experiments where adsorption was of minor relevance before. Before starting the desorption experiments the aqueous medium, containing still a considerable amount of test item in cases where adsorption is not relevant, has to be replaced by fresh aqueous medium. One can imagine that the replacement is not quantitative because minor amounts of the aqueous medium and, therefore also test item, remain attached to the soil surface. These residues becomes significant in these cases leading to implausible amounts for desorption. 

% of Desorption Coefficients for HEBMP Cyclic Form, pH-neutral in Eurosoils

Desorption experiments lasted 24h.

Application concentration prior to desorption experiments was 10 mg HEBMP Cyclic Form, pH-neutral/L.

Replicate	Eurosoils
	1	2	3	4	5
	% of Desorption
1	22	40	36	n.c.	n.c.
2	23	60	48	n.c.	n.c.

Reference 2

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-02 to 2013-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline with appropriate methods, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The study was based on these guidelines. Adaptions were necessary due to the test item specific physico-chemical properties.

GLP compliance:

yes

Type of method:

batch equilibrium method

Media:

soil

Radiolabelling:

no

Test temperature:

21+-2°C; Minimum: 18.9°C, Maximum: 21.7°C

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 30 mg/L for adsorption kinetics, impact of Ca2+ and desorption additional 150 and 300 mg/L for concentration dependency
(adsorption isotherms)
- Stock solutions: Stock solutions of 3 g and 30 g test item/L were prepared in HPLC water
- Replicates and sampling intervals:
Replicates (adsorption kinetics): Duplicates per sampling point for the adsorption kinetics. Duplicate samples were agitated for 1, 2, 4, 6, 24 and
30 h. 0.4 mL of the stock solution (3 g/L) were added to the test systems consisting of 1 g soil and 39.6 mL aqueous medium.
Replicates (desorption): The replicates of the adsorption kinetics which had been agitated for 24h were used for this purpose. Therefore, the
aqueous medium was decanted into a graduated cylinder after centrifugation and the volume was determined. After that fresh aqueous medium
was added to the test systems to a final volume of 40 mL. The samples were agitated for 24h.
Replicates (adsorption isotherms): Duplicate samples were agitated for 24h. Aliquots (0.2 and 0.4 mL) from the stock solution with a
concentration of 20 g test item/L were used for fortification. In addition, 0h samples were prepared as reference by spiking 0.05 and 0.1 mL of
the stock solution into 9.95 and 9.9 mL aqueous medium, respectively.
Replicates (Ca2+ dependency): Duplicate samples were prepared by adding 0.4 mL of the stock solution with a concentration of 2 g test item/L in
to aqueous media with concentrations of 40 and 400 mg CaCl2/L. These samples were agitated for 24h. 0h samples in these aqueous media were
prepared by spiking 29.7 mL with 0.3 mL of the stock solution with a concentration of 2 g/L.

No precipitation after addition of the test item to the equilibrated aqueous media, which contained the corresponding concentrations of CaCl2,
was observed. Furthermore no precipitated phase of the test item after agitation of these samples was observed during the study.

TEST ITEM CONTROLS (adsorption kinetics): Furthermore test item controls (duplicates) were prepared to investigate the test item stability during
agitation on the overhead shaker. Therefore the supernatant was removed and fortified to a concentration of 30 mg test item/L. An aliquot of
these samples was used for the determination of the reference value (0h value) via photometry. The remaining part of the samples was agitated
for 30h.

Blank samples for the adsorption kinetics and the investigations about concentration dependency were prepared as described for the test item
replicates but without fortification with the test item. Blank samples for experiments about Ca2+ dependency were obtained from the 0h samples
prior to fortification with the test item. Blank samples for desorption experiments were obtained from a blank sample of the adsorption kinetic
experiments which had been treated and agitated analogously to the corresponding test item replicates. Aliquots of these samples were analysed
for background correction during photometric analysis.

- Sample storage before analysis: at room temperature

Details on matrix:

Standard eurosoils no. 1, 2, 3, 4 and 5 in contact to demineralised water were used for this study. These soils have varying adsorption capacities in relation to their content of organic matter, clay and metal oxides as well as pH and cation exchange capacity. All soils were air dried and sieved to a maximum particle size of 2 mm. The physico-chemical characteristics of the different soils are given in the table below.

Soil Characteristics of Eurosoils :
Eurosoils
1 2 3 4 5
Soil site 1) Sicily Peleponnes Wales Normandy Schleswig-Holstein
(Italy) (Greece) (Great Britain) (France) (Germany)
FAO soil unit 1) Vertic Rendzina Dystric Orthic Orthic
Cambisol Cambisol Luvizol Podzol
pH 1) 6.2 8.1 6.2 7.5 4.1
Total Carbon [%] 1) 3.39 10.81 3.25 1.45 6.43
Organic Carbon [%] 1) 3.27 3.72 3.01 1.31 5.96
Nitrogen [%] 1) 0.34 0.25 0.31 0.16 0.23
Dry mass [%] 2) 94.5 97.2 98.0 97.3 98.4
Clay (<0.002 mm) 3) 74.6 29.0 17.3 20.9 4.3
Silt (0.002-0.063 mm) 3) 24.5 56.1 32.8 73.2 8.8
Sand (0.063-2 mm) 3) 1.0 15.0 50.0 6.0 87.0

1) Gawlik and Muntau, Eurosoils II Laboratory and Reference Materials for Soil-related Studies, Environment Institute 1999
2) determined at DR.U.NOACK-LABORATORIEN
3) determined at INSTITUT KOLDINGEN AGROLAB LABORGRUPPE (non-GLP), mean value of two replicates

Details on test conditions:

TEST CONDITIONS
- pH: the pH values of the aqueous media of the test systems were measured before and after equilibration with the corresponding soils as well as
after addition of the test item. The pH values ranged between 4.62 and 7.85 after incubation with the soils and from 4.47 to 7.58 after addition of the
test item. Among the soils used for the experiment, eurosoil 5 was the most acidic soil, while the most basic soil was eurosoil 2. The test item,
HEBMP linear form, pH-neutral, did not have a significant impact on the pH value of the test system.

pH values of the aqueous Media:
(Values before and after soil contact as well as after addition of the test item are given. Mean values of two replicates are described.)

Eurosoils
1 2 3 4 5
before 6.02 6.02 6.22 6.18 6.00
after soil contact 5.97 7.85 6.35 7.33 4.62
after addition of the test ite 6.08 7.58 6.41 7.18 4.47


TEST SYSTEM
- Type, size and further details on reaction vessel: 50 mL centrifugation tubes, VWR
- Soil / Solution ratio:1 g soil and 40 mL solution
- Aqueous media: Tap water demineralised by reverse osmosis, additional 40 and 400 mg/L CaCl2 for investigation of impact of Ca2+ on adsorption
- Agitation / Test temperature: By overhead shaker at 21 +- 2 °C; Frequency was adjusted to avoid sedimentation of soil particles during treatment.
- Equilibration: The test systems were equilibrated with aqueous medium by agitation overnight (minimum 12h).
- Sample preparation: Equilibrated replicates were centrifugated (10 min. at 10000 g) prior to fortification. Prior to photometrical analysis replicates
were centrifugated (10 min. at 10000 g) and filtrated (0.45 µm).
- Measuring equipment: Photometer Nanocolor UV / Vis, MACHEREY & NAGEL
- Are the residues from the adsorption phase used for desorption: yes

Computational methods:

Determination of adsorption kinetics, % of adsorption, adsorption isotherms and % of desorption was performed according to the equations given
below.
These endpoints were determined by the indirect method. Only the test item concentrations in the aqueous media were monitored. Decline of the test item in the aqueous media by other reasons than adsorption on soil could be ruled out as demonstrated by preliminary experiments
(Tier 1, non-GLP). Results of test item controls during these experiments with eurosoils 1 and 5 showed that adsorption on test vessels did not occur.
In addition, test item controls were prepared for all soils and treated as the test item replicates during experiments for determination of adsorption
kinetics in the definitive study.


% Adsorption: [%] Adsorption = (m0 - madsaq(ti)) / m0 * 100

m0 = initial nominal mass of test item in aqueous phase [mg]
madsaq(ti) = mass of test item measured in the aqueous phase at the point of time ti [mg]


Distribution Coefficient KD (Adsorption):

KD = (madss(eq)/madsaq(eq)) * (Vaq/msoil) [mL/g]

madss = mass of test item in the soil phase at equilibrium [mg]
madsaq = mass of test item in the aqueous phase at equilibrium [mg]
m soil = used amount of soil [g]
V aq = used volume of aqueous phase [mL]


Adsorption Isotherms:
The adsorbed mass per unit mass of soil was plotted as a function of the calculated equilibrium concentration of the test item. The FREUNDLICH
adsorption isotherm equation relates the amount of the test item adsorbed to the concentration of the test item in solution at equilibrium.

Cadss (eq) = KadsF * Cadsaq (eq)^(1/n)

Cadss (eq) = concentration of the test item adsorbed on the soil at adsorption equilibrium [mg/g]
KadsF = Freundlich adsorption coefficient mg^(1-1/n)(mL)^(1/n) g^-1]
Cadsaq (eq) = concentration of the test item in the aqueous phase at adsorption equilibrium [mg/mL]
1/n = regression constant

In its linear form the FREUNDLICH adsorption equation is as follows:

log Cadss (eq) = log KadsF + (1/n) * log Cadsaq (eq)

The reciprocal regression coefficient was directly deduced from the slope of the linear regression if sufficient linearity was obtained.



% Desorption:
[%] Desorption = (mdesaq / (m0 - madsaq)) * 100%

m0 = initial nominal mass of test item [mg]
madsaq = mass of test item measured in the aqueous phase after the adsorption step [mg]
mdesaq = mass of test item measured in the aqueous phase after the desorption step [mg]


Software:
The data for the tables in the report were computer generated (EXCEL, MICROSOFT CORPORATION) and rounded for presentation from the full
derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Type:

Kd

Value:

393 L/kg

% Org. carbon:

3.27

Remarks on result:

other: Eurosoil 1; temperature: 18.9°C to 21.7°C.

Type:

Kd

Value:

186 L/kg

% Org. carbon:

3.72

Remarks on result:

other: Eurosoil 2; temperature: 18.9°C to 21.7°C

Type:

Kd

Value:

28 L/kg

% Org. carbon:

3.01

Remarks on result:

other: Eurosoil 3; temperature: 18.9°C to 21.7°C

Type:

Kd

Value:

146 L/kg

% Org. carbon:

1.31

Remarks on result:

other: Eurosoil 4; temperature: 18.9°C to 21.7°C

Type:

Kd

Value:

34 L/kg

% Org. carbon:

5.96

Remarks on result:

other: Eurosoil 5; temperature: 18.9°C to 21.7°C

Type:

Kd

Value:

> 3 953 L/kg

% Org. carbon:

3.27

Remarks on result:

other: Eurosoil 1; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 278 - <= 324 L/kg

% Org. carbon:

3.72

Remarks on result:

other: Eurosoil 2; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 383 - <= 422 L/kg

% Org. carbon:

3.01

Remarks on result:

other: Eurosoil 3; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 261 - <= 271 L/kg

% Org. carbon:

1.31

Remarks on result:

other: Eurosoil 4; CaCl2 present in aqueous phase at 400 mg/L

Type:

Kd

Value:

>= 245 - <= 250

% Org. carbon:

5.96

Remarks on result:

other: Eurosoil 5; CaCl2 present in aqueous phase at 400 mg/L

Recovery of test material:

not applicable. HEBMP could not be analysed in the solid phase due to effectively irreversible binding.

Transformation products:

not measured

Details on results (Batch equilibrium method):

- Eurosoil 1:
Adsorption kinetics: Adsorption was complete after 2h of agitation. 91% of the initial mass of the test item in the aqueous phase were adsorbed in
this period. 90% adsorption were determined at the end of the agitation period (30h). Based on the mean value for the adsorbed fractions (1 – 30h)
a distribution coefficient KD of 393 mL/g was calculated. The degree of adsorption [%] was calculated according to equation (1) and value for the
distribution coefficient KD according to equation (2). The mean recovery of 101% for the test item controls indicated that test item adsorption on
the test vessels did not occur and only adsorption on soil was responsible for the decline of the test item in the aqueous phase.
Adsorption isotherms: The adsorbed amount of the test item on soil increased with higher loading level of the test item in the aqueous medium.
Nevertheless the adsorption behaviour was not linear and the distribution coefficients KD decreased from a maximum of 463 mL/g at 30 mg test
item/L to 33 mL/g at a concentration of 300 mg test item/L. The graphical expression of the adsorption behaviour was characterised by a
coefficient of determination R2 of 0.995. The regression line showed linearity and the reciprocal regression coefficient 1/n was 0.3832 indicating
that adsorption behaviour was not linear in the investigated concentration range.
Impact of Ca2+ on adsorption: The results demonstrate the increase of the distribution coefficient KD with increasing Ca2+ concentrations though
already a high degree of adsorption with 91 – 92% without addition of exogenous Ca2+ was obtained. Distribution coefficients KD from maximum
463 mL/g in demineralised water steadily increased to a value for KD > 3953 mL/g when a 400 mg/L solution of CaCl2 as aqueous medium was
used for the adsorption experiment.
Desorption: the desorbed amount of the test item was not higher than 3%.
- Eurosoil 2:
Adsorption Kinetics: % adsorption were 81 and 83% for the two replicates after 30h of agitation. Adsorption increased up to 72% after 1h of agitation
and up to 80% after 24h of agitation. Based on the mean value for the adsorbed fraction (82%) after 30h a distribution coefficient KD of 186 mL/g
was calculated. The mean recovery of 103% for the test item controls indicated that test item adsorption on the test vessels did not occur and only
adsorption on soil was responsible for the decline of the test item in the aqueous phase.
Adsorption isotherms: the distribution coefficients KD decreased from a maximum of 165 mL/g at 30 mg test item/L to minimum 34 mL/g at a
concentration of 300 mg test item/L indicating no linear adsorption behaviour in the investigated concentration range. No regression coefficient
1/n was determined because no linear regression line was obtained.
Impact of Ca2+ on adsorption: the results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item in this soil.
An increase of the distribution coefficient KD from maximum 165 mL/g in demineralised water to a maximum value for KD of 324 mL/g for the
treatment with 400 mg/L CaCl2 was determined.
Desorption: 11% of the adsorbed mass were desorbed over a period of 24h.
- Eurosoil 3:
Adsorption kinetics: % adsorption were 25 and 26% for the two replicates after 1h of agitation and increased to 40 and 43% after 30h of agitation. No
significant increase in adsorption was observed after 24h of agitation. Therefore, based on the mean value of adsorption for the 24h and 30h
samples a distribution coefficient KD of 28 mL/g was calculated. The mean recovery of 97% for the test item controls indicated that test item
adsorption on the test vessels did not occur and only adsorption on soil was responsible for the decline of the test item in the aqueous phase.
Adsorption isotherms: The distribution coefficients KD decreased from a maximum of 27 mL/g at 30 mg test item/L to minimum 5 mL/g at the
concentration levels 150 and 300 mg test item/L. No linear adsorption behaviour in the investigated concentration range was observed. No
regression coefficient 1/n was determined because no linear regression line was obtained.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item in this soil. An
increase of the distribution coefficient KD from maximum 27 mL/g in demineralised water to a maximum value for KD of 422 mL/g for the
treatment with 400 mg/L CaCl2 was determined.
Desorption: Up to 17% of the adsorbed mass were desorbed over a period of 24h.
- Eurosoil 4:
Adsorption kinetics: % adsorption were between 77 and 79% in equilibrium. Equilibrium was reached after 4h of agitation. Based on the mean value
for the adsorbed fraction (from 4h to 30h of agitation) a distribution coefficient KD of 146 mL/g was calculated. The mean recovery of 101% for the
test item controls indicated that test item adsorption on the test vessels did not occur and only adsorption on soil was responsible for the decline of
the test item in the aqueous phase.
Adsorption isotherms: The distribution coefficients KD decreased from a maximum of 151 mL/g at 30 mg test item/L to 15 mL/g at a concentration
of 300 mg test item/L indicating no linear adsorption behaviour in the investigated concentration range. The regression analysis showed linearity.
The regression coefficient 1/n was 0.3501 and the coefficient of determination R2 was 0.9994.
Impact of Ca2+ on adsorption: The results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item in this soil. An
increase of the distribution coefficient KD from maximum 151 mL/g in demineralised water to a maximum value for KD of 271 mL/g for the
treatment with 400 mg/L CaCl2 was observed.
Desorption: Only up to 9% of the adsorbed fraction desorbed over a period of 24h.
- Eurosoil 5:
Adsorption kinetics: % adsorption were between 9 and 10% after 1h of agitation. Adsorption further increased and equilibrium was reached after
24h of agitation with adsorption between 42 and 49%. Based on the mean value for the adsorbed fraction (from 24h to 30h of agitation) a
distribution coefficient KD of 34 mL/g was calculated. The mean recovery of 102% for the test item controls indicated that test item adsorption on
the test vessels did not occur and only adsorption on soil was responsible for the decline of the test item in the aqueous phase.
Adsorption isotherms: The distribution coefficients KD decreased from a maximum of 39 mL/g at 30 mg test item/L to 2 mL/g at a concentration of
300 mg test item/L indicating no linear adsorption behaviour in the investigated concentration range. No regression coefficient 1/n was calculated
because no linear regression line was obtained.
Impact of Ca2+ on adsorption: the results indicate that Ca2+ in the aqueous medium enhances the adsorption of the test item in this soil. An
increase of the distribution coefficient KD from maximum 39 mL/g in demineralised water to a maximum value for KD of 250 mL/g for the
treatment with 400 mg/L CaCl2 was observed.
Desorption: the measured amount of the test item was below the working range of the photometric test 0-761.

Tables:

Eurosoil 1 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 5.00 1.200 0.000 0
2 0 5.27 1.200 0.000 0
1 1 0.48 0.112 1.088 91
2 1 0.48 0.112 1.088 91
1 2 0.56 0.131 1.069 89
2 2 0.54 0.126 1.074 89
1 4 0.51 0.119 1.081 90
2 4 0.53 0.124 1.076 90
1 6 0.51 0.119 1.081 90
2 6 0.49 0.115 1.085 90
1 24 0.43 0.100 1.100 92
2 24 0.46 0.107 1.093 91
1 30 0.49 0.115 1.085 90
2 30 0.51 0.119 1.081 90


Eurosoil 1 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

[mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
30 0 1 5.00 1.200 0.000 - - - -
0 2 5.27 1.200 0.000 - - - -
24 1 0.43 0.100 1.100 -2.600 0.066 92 463
24 2 0.46 0.107 1.093 -2.571 0.063 91 430
150 0 1 23.54 6.000 0.000 - - - -
0 2 23.95 6.000 0.000 - - - -
24 1 8.64 2.183 3.817 -1.263 0.606 64 74
24 2 8.64 2.183 3.817 -1.263 0.606 64 74
300 0 1 49.38 12.000 0.000 - - - -
0 2 48.56 12.000 0.000 - - - -
24 1 27.65 6.776 5.224 -0.771 0.743 44 33
24 2 27.32 6.695 5.305 -0.776 0.749 44 34



Eurosoil 1 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 5.00 1.200 0.000 - -
Ca2+2 2 0 5.27 1.200 0.000 - -
1 24 0.43 0.100 1.100 92 463
2 24 0.46 0.107 1.093 91 430
40 mg/L
CaCl2
1 0 4.77 1.200 0.000 - -
2 0 4.73 1.200 0.000 - -
1 24 0.09 0.023 1.177 98 2192
2 24 0.09 0.023 1.177 98 2192
400 mg/L
CaCl2
1 0 4.70 1.200 0.000 - -
2 0 4.74 1.200 0.000 - -
1 24 < 0.05(1) < 0.013 > 1.187 99 > 3953
2 24 < 0.05(1) < 0.013 > 1.187 99 > 3953


(1) measured conc. < lower limit working range of test 0-761(0.05 – 1.5 mg total phosphorus/L)

Eurosoil 1 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 5.00 1.200 0.000 - -
2 0 5.27 1.200 0.000 - -
1 24h Adsorption 0.43 0.100 1.100 92 -
2 24h Adsorption 0.46 0.107 1.093 91 -
1 24h Desorption 0.12 0.028 1.071 - 3
2 24h Desorption 0.12 0.028 1.064 - 3


Eurosoil 2 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 4.74 1.200 0.000 0
2 0 4.79 1.200 0.000 0
1 1 1.40 0.353 0.847 71
2 1 1.35 0.340 0.860 72
1 2 1.25 0.315 0.885 74
2 2 1.25 0.315 0.885 74
1 4 1.05 0.264 0.936 78
2 4 1.04 0.262 0.938 78
1 6 1.17 0.295 0.905 75
2 6 1.12 0.282 0.918 76
1 24 0.95 0.239 0.961 80
2 24 0.95 0.239 0.961 80
1 30 0.81 0.204 0.996 83
2 30 0.92 0.232 0.968 81


Eurosoil 2 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
30 0 1 4.74 1.200 0.000 - - - -
0 2 4.79 1.200 0.000 - - - -
24 1 0.95 0.239 0.961 -2.223 -0.005 80 165
24 2 0.95 0.239 0.961 -2.223 -0.005 80 165
150 0 1 24.03 6.000 0.000 - - - -
0 2 24.53 6.000 0.000 - - - -
24 1 12.10 2.990 3.010 -1.126 0.491 50 41
24 2 12.34 3.049 2.951 -1.118 0.482 49 40
300 0 1 50.04 12.000 0.000 - - - -
0 2 51.52 12.000 0.000 - - - -
24 1 27.82 6.574 5.426 -0.784 0.747 45 34
24 2 24.36 5.757 6.243 -0.842 0.808 52 45


Eurosoil 2 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]

without
exogenous 1 0 4.74 1.200 0.000 - -
Ca2+ 2 0 4.79 1.200 0.000 - -
1 24 0.95 0.239 0.961 80 165
2 24 0.95 0.239 0.961 80 165
40 mg/L
CaCl2
1 0 4.97 1.200 0.000 - -
2 0 5.00 1.200 0.000 - -
1 24 0.86 0.207 0.993 83 197
2 24 0.94 0.226 0.974 81 177
400 mg/L
CaCl2
1 0 5.02 1.200 0.000 - -
2 0 4.91 1.200 0.000 - -
1 24 0.64 0.155 1.045 87 278
2 24 0.56 0.135 1.065 89 324


Eurosoil 2 – Desorption
Determination of the desorbed fractions after 24h adsorption and 24h desorption.

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 4.74 1.200 0.000 - -
2 0 4.79 1.200 0.000 - -
1 24h Adsorption 0.95 0.239 0.961 80 -
2 24h Adsorption 0.95 0.239 0.961 80 -
1 24h Desorption 0.42 0.106 0.855 - 11
2 24h Desorption 0.42 0.106 0.855 - 11


Eurosoil 3 – Adsorption Kinetics
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 5.17 1.200 0.000 0
2 0 5.07 1.200 0.000 0
1 1 3.85 0.902 0.298 25
2 1 3.79 0.888 0.312 26
1 2 3.60 0.844 0.356 30
2 2 3.62 0.848 0.352 29
1 4 3.60 0.844 0.356 30
2 4 3.56 0.834 0.366 30
1 6 3.47 0.813 0.387 32
2 6 3.44 0.806 0.394 33
1 24 3.08 0.722 0.478 40
2 24 3.11 0.729 0.471 39
1 30 3.05 0.715 0.485 40
2 30 2.93 0.687 0.513 43


Eurosoil 3 - Adsorption Isotherms
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]

30 0 1 5.17 1.200 0.000 - - - -
0 2 5.07 1.200 0.000 - - - -
24 1 3.08 0.722 0.478 -1.744 -0.312 40 27
24 2 3.11 0.729 0.471 -1.739 -0.318 39 26
150 0 1 25.10 6.000 0.000 - - - -
0 2 25.60 6.000 0.000 - - - -
24 1 22.55 5.337 0.663 -0.875 -0.170 11 5
24 2 22.22 5.259 0.741 -0.881 -0.122 12 6
300 0 1 53.49 12.000 0.000 - - - -
0 2 52.34 12.000 0.000 - - - -
24 1 47.57 10.788 1.212 -0.569 0.092 10 5
24 2 47.40 10.749 1.251 -0.571 0.106 10 5



Eurosoil 3 – Impact of Ca2+ on Adsorption
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 5.17 1.200 0.000 - -
Ca2+ 2 0 5.07 1.200 0.000 - -
1 24 3.08 0.722 0.478 40 27
2 24 3.11 0.729 0.471 39 26
40 mg/L
CaCl2
1 0 4.74 1.200 0.000 - -
2 0 5.20 1.200 0.000 - -
1 24 1.93 0.466 0.734 61 64
2 24 1.93 0.466 0.734 61 64
400 mg/L
CaCl2
1 0 4.97 1.200 0.000 - -
2 0 5.00 1.200 0.000 - -
1 24 0.48 0.116 1.084 90 383
2 24 0.44 0.106 1.094 91 422


Eurosoil 3 – Desorption
(Determination of the desorbed fractions after 24h adsorption and 24h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 5.17 1.200 0.000 - -
2 0 5.07 1.200 0.000 - -
1 24h Adsorption 3.08 0.722 0.478 40 -
2 24h Adsorption 3.11 0.729 0.471 39 -
1 24h Desorption 0.29 0.068 0.410 - 14
2 24h Desorption 0.34 0.080 0.391 - 17


Eurosoil 4 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 5.14 1.200 0.000 0
2 0 5.04 1.200 0.000 0
1 1 1.33 0.314 0.886 74
2 1 1.35 0.318 0.882 73
1 2 1.22 0.288 0.912 76
2 2 1.22 0.288 0.912 76
1 4 1.09 0.257 0.943 79
2 4 1.14 0.269 0.931 78
1 6 1.19 0.281 0.919 77
2 6 1.15 0.271 0.929 77
1 24 1.12 0.264 0.936 78
2 24 1.09 0.257 0.943 79
1 30 1.10 0.259 0.941 78
2 30 1.07 0.252 0.948 79


Eurosoil 4 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
30 0 1 5.14 1.200 0.000 - - - -
0 2 5.04 1.200 0.000 - - - -
24 1 1.12 0.264 0.936 -2.180 -0.017 78 146
24 2 1.09 0.257 0.943 -2.192 -0.014 79 151
150 0 1 25.02 6.000 0.000 - - - -
0 2 25.18 6.000 0.000 - - - -
24 1 15.06 3.600 2.400 -1.046 0.392 40 27
24 2 15.06 3.600 2.400 -1.046 0.392 40 27
300 0 1 50.53 12.000 0.000 - - - -
0 2 48.89 12.000 0.000 - - - -
24 1 36.38 8.782 3.218 -0.658 0.519 27 15
24 2 36.54 8.821 3.179 -0.657 0.514 26 15



Eurosoil 4 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without exogenous
exogenous 1 0 5.14 1.200 0.000 - -
Ca2+ 2 0 5.04 1.200 0.000 - -
1 24 1.12 0.264 0.936 78 146
2 24 1.09 0.257 0.943 79 151
40 mg/L
CaCl2
1 0 4.94 1.200 0.000 - -
2 0 4.87 1.200 0.000 - -
1 24 0.91 0.223 0.977 81 180
2 24 0.94 0.230 0.970 81 173
400 mg/L
CaCl2
1 0 4.79 1.200 0.000 - -
2 0 4.92 1.200 0.000 - -
1 24 0.64 0.158 1.042 87 271
2 24 0.66 0.163 1.037 86 261


Eurosoil 4 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24 h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 5.14 1.200 0.000 - -
2 0 5.04 1.200 0.000 - -
1 24h Adsorption 1.12 0.264 0.936 78 -
2 24h Adsorption 1.09 0.257 0.943 79 -
1 24h Desorption 0.29 0.068 0.868 - 7
2 24h Desorption 0.34 0.080 0.863 - 9


Eurosoil 5 – Adsorption Kinetics:
(The initial measured value in [mg P/L] was set to the initial amount maq [mg] in the aqueous medium. Values are background corrected.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%]
1 0 4.97 1.200 0.000 0
2 0 5.12 1.200 0.000 0
1 1 4.53 1.078 0.122 10
2 1 4.61 1.097 0.103 9
1 2 4.54 1.080 0.120 10
2 2 4.43 1.054 0.146 12
1 4 4.07 0.968 0.232 19
2 4 4.00 0.951 0.249 21
1 6 3.97 0.944 0.256 21
2 6 3.39 0.806 0.394 33
1 24 2.91 0.692 0.508 42
2 24 2.57 0.611 0.589 49
1 30 2.63 0.626 0.574 48
2 30 2.81 0.668 0.532 44


Eurosoil 5 - Adsorption Isotherms:
(Volume of the aqueous medium in [mL] and soil dry weight in [g] were taken into account for calculations. Concentrations in aqueous medium and
soil after 24h were considered.)

c [mg/L] t [h] Replicate cmeasured [mg P/L] m aq [mg] m soil [mg] log c aq log c soil Adsorption [%] KD [mL/g]
30 0 1 4.97 1.200 0.000 - - - -
0 2 5.12 1.200 0.000 - - - -
24 1 2.91 0.692 0.508 -1.762 -0.287 42 30
24 2 2.57 0.611 0.589 -1.816 -0.223 49 39
150 0 1 24.11 6.000 0.000 - - - -
0 2 23.87 6.000 0.000 - - - -
24 1 21.48 5.372 0.628 -0.872 -0.195 10 5
24 2 20.82 5.207 0.793 -0.885 -0.094 13 6
300 0 1 47.40 12.000 0.000 - - - -
0 2 48.06 12.000 0.000 - - - -
24 1 45.43 11.422 0.578 -0.544 -0.231 5 2
24 2 44.94 11.299 0.701 -0.549 -0.147 6 3


Eurosoil 5 – Impact of Ca2+ on Adsorption:
(Values were calculated after 24h of agitation.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] KD [mL/g]
without
exogenous 1 0 4.97 1.200 0.000 - -
Ca2+ 2 0 5.12 1.200 0.000 - -
1 24 2.91 0.692 0.508 42 30
2 24 2.57 0.611 0.589 49 39
40 mg/L
CaCl2
1 0 4.77 1.200 0.000 - -
2 0 5.09 1.200 0.000 - -
1 24 1.12 0.273 0.927 77 138
2 24 1.04 0.253 0.947 79 152
400 mg/L
CaCl2
1 0 4.53 1.200 0.000 - -
2 0 4.48 1.200 0.000 - -
1 24 0.63 0.168 1.032 86 250
2 24 0.64 0.170 1.030 86 245


Eurosoil 5 – Desorption:
(Determination of the desorbed fractions after 24h adsorption and 24 h desorption.)

Replicate t [h] cmeasured [mg P/L] m aq [mg] m soil [mg] Adsorption [%] Desorption [%]
1 0 4.97 1.200 0.000 - -
2 0 5.12 1.200 0.000 - -
1 24h Adsorption 2.91 0.692 0.508 42 -
2 24h Adsorption 2.57 0.611 0.589 49 -
1 24h Desorption < 0.05(1) < 0.012 > 0.496 - < 2
2 24h Desorption < 0.05(1) < 0.012 > 0.577 - < 2

(1) measured conc. < lower limit of test 0-761 (0.05 – 1.5 mg total phosphorus /L)

Validity criteria fulfilled:

yes

Conclusions:

The conclusions drawn from all experiments are as follows:

1) The degree of adsorption for HEBMP linear form, pH-neutral correlates with the clay and silt amount of the tested soils for eurosoils 1, 2 and 4. Higher
distribution coefficients KD for the test item result in soils with higher amounts of clay and silt. The impact of the clay fraction dominates about the impact
of the silt fraction. The test item has lower distribution coefficients KD in eurosoils 3 and 5 as expected because these soils have the lowest amounts of clay
and silt. But the test item has a higher KD in eurosoil 5 than in eurosoil 3 though amount of clay and silt are lower. Therefore, one can assume that also
other soil fractions can contribute to the adsorption of the test item (e.g. sand or organic carbon).

2) No linear adsorption behaviour was observed in the investigated concentration range (30 – 300 mg/L) for HEBMP linear form, pH-neutral in eurosoil 1 – 5.

3) Ca2+ in the aqueous medium of the adsorption experiments enhances the adsorption behaviour of HEBMP linear form, pH-neutral.

4) % of Desorption ranges between < 2 and 17% for HEBMP linear form, pH-neutral in the investigated eurosoils indicating that desorption is only of minor
relevance for the test item.

Executive summary:

The adsorption / desorption behaviour of HEBMP linear form, pH-neutral (batch no.SK426) in 5 different soils was determined following OECD

guideline 106 and EC C.18. The study was conducted from 2012-11-02 to 2013-05-03, at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

The test item was a dialkylphosphonohydroxyethyl amine derivative.

Adsorption Kinetics in Eurosoils:

The table below shows the results obtained for the determination of distribution coefficients K_D in eurosoils 1 to 5. Adsorption experiments lasted 30h. The results demonstrate under consideration of the a.m. soil parameters that there is a positive correlation between the degree of adsorption at equilibrium and the proportion of soil components with smaller particle sizes (especially the clay fraction) in the soils. The highest K_D with

393 mL/g was obtained in eurosoil 1. This soil is characterised by the highest clay content of 74.6% in all investigated eurosoils. Silt and sand are

minor constituents of this soil at 24.5 and 1.0% w/w respectively. It follows eurosoil 2 (K_D 186 mL/g, ca. 85% clay + silt, of which 29% clay) and eurosoil 4 (K_D 146 mL/g, ca. 94% clay + silt, of which 21% clay). A slight higher K_D with 34 mL/g was observed in eurosoil 5 (13% clay + silt) compared to a K_D with 28 mL/g in eurosoil 3 (ca. 50% clay + silt) though the amount of clay and silt was lower. This fact could indicate that further

soil characteristics can have an impact on the adsorption behaviour of the test item. Eurosoil 5 has the highest amount of sand (87%) and organic carbon (6.0%) for all investigated eurosoils. All these results demonstrate that the clay fraction has the highest impact on the adsorption of the test item followed by the silt and sand or organic carbon.

Distribution Coefficients K_D for HEBMP linear form, pH-neutral in Eurosoils:

Adsorption experiments lasted 30h.

Application concentration 30 mg HEBMP linearform, pH-neutral/L

	Eurosoils
	1	2	3	4	5
KD[mL/g]	393	186	28	146	34

Adsorption Isotherms in Eurosoils:

The table below shows the results for the concentration dependency of adsorption. Therefore, additional concentration levels of 150 and

300 mg test item/L were tested, the degree of adsorption as mass adsorbed on soil was determined after 24h of agitation and compared with the values obtained in the lowest application concentration with 30 mg test item/L. A steady increase in the amount adsorbed on soil was determined for eurosoils 1 - 4. In contrast, no clear tendency for the results in eurosoil 5 was observed. Similar results were obtained in eurosoil 3 and 5 when the loading level was increased to 150 mg test item/L. But, no more mass was adsorbed in eurosoil 5 when the concentration was doubled to

300 mg test item/L whereas an increase in eurosoil 3 was still visible. Probably, other soil fractions than clay and silt can adsorb the test item in eurosoil 5 and compensate for the low amount of clay and silt up to a threshold concentration. Above this concentration the capacity of this soil is exhausted. Regression coefficients 1/n were only calculated for eurosoils 1 and 4 because the regression lines of the graphs were linear. No regression coefficients 1/n were calculated for the other eurosoils. No linearity was obtained during the corresponding regression analysis.

Adsorbed Mass on Soils in Dependence on Test Item Concentration:

Samples were agitated for 24h.

Total amount of test item applied per replicate in the different concentration levels: 1.2, 6, 12 [mg]

Test Item Concentration [mg/L]	Replicate	Eurosoils
		1	2	3	4	5
		msoil[mg]
30	1	1.100	0.961	0.478	0.936	0.508
	2	1.093	0.961	0.471	0.943	0.589
150	1	3.817	3.010	0.663	2.400	0.628
	2	3.817	2.951	0.741	2.400	0.793
300	1	5.224	5.426	1.212	3.218	0.578
	2	5.305	6.243	1.251	3.179	0.701
R^2		0.9950	0.9659	0.8606	0.9994	0.3135
Linearity		yes	no	no	yes	no
1/n		0.3832	n.d.	n.d.	0.3501	n.d.

n.d. = could not be determined due to no linearity of the corresponding regression line.

Eurosoils – Impact of Ca²⁺ on Adsorption:

 The table below summarises the values for the distribution coefficients K_D of HEBMP linear form, pH-neutral in the different eurosoils in dependence on increasing concentration of exogenous Ca²⁺ in the aqueous medium. Therefore, further adsorption experiments were performed with concentration of 40 and 400 mg/L CaCl₂ in the aqueous medium and the resulting distribution coefficients K_D were compared with those

obtained without exogenous Ca²⁺. The samples were agitated for 24h. The data show that exogenous Ca²⁺ enhances the adsorption of the test

item in all eurosoils. A steady increase of the adsorbed mass was observed with increasing concentrations of Ca²⁺. The impact of Ca²⁺on the

adsorption of the test item on soil was emphasised by the fact that the increase in adsorption was significantly more pronounced in eurosoils

3 and 5 than in eurosoil 2 and 4 though containing lower amounts of silt and clay. When the exogenous Ca²⁺ concentration was enhanced to

400 mg/L CaCl₂ the differences in the corresponding distribution coefficients K_D became lower in eurosoils 2 - 5 with the highest K_D now in eurosoil 3 (up to 422 mL/g). These data let assume that in the presence of Ca²⁺ also the sand fraction has an important impact on the adsorption

behaviour of the test item.

Distribution Coefficients K_D for HEBMP linear form, pH-neutral in Eurosoils in Dependence on exogenous Ca²⁺Concentrations:

Adsorption experiments lasted 24h.

Application concentration 30 mg HEBMP linearform, pH-neutral/L

 

Concentration exogenous CaCl2 [mg/L]	Replicate	Eurosoils
		1	2	3	4	5
		KD [mL/g]
0	1	463	165	27	146	30
	2	430	165	26	151	39
40	1	2192	197	64	180	138
	2	2192	177	64	173	152
400	1	> 3953	278	383	271	250
	2	> 3953	324	422	261	245

¹⁾        measured conc. < lower limit working range of photometric test

Eurosoils – Desorption:

The table below shows the results of the desorption experiments. Desorption experiments were performed after an adsorption period of 24h and lasted 24h. The data indicate that desorption is of minor relevance for the test item. Desorption was not higher than 17%.

% of Desorption for HEBMP linear form, pH-neutralin Eurosoils:

Desorption experiments lasted 24h.

Application concentration prior to desorption experiments was 30 mg HEBMP linear form, pH-neutral/L.

Replicate	Eurosoils
	1	2	3	4	5
	% of Desorption
1	3	11	14	7	< 2
2	3	11	17	9	< 2

Description of key information

Adsorption coefficients have been derived for HEBMP-xNa in two reliable OECD 106 tests in soil-water systems under a range of conditions. At low concentrations of the range predicted to occur in the environment, the average Kd has been extrapolated to 1500 l/kg (for the linear constituent structure). There is some evidence that binding in high-clay substrates is likely to be higher still and in hard waters (calcium concentration equivalent to 40 mg/l or higher) the Kd would be significantly higher. For the cyclic constituent, the equivalent value was 110 l/kg based on a preliminary test at higher concentration.

Adsorption and desorption behaviour of the cyclic constituent is not yet fully understood and until/unless definitively proven otherwise, the chemical safety assessment assumes at this stage that some desorption of cyclic constituent from the substrate is possible. This is a conservative interpretation and is not the case for the linear constituent (and other aminomethylenephosphonates) which show negligible desorption and effectively irreversible binding. An explanation of the values used for calculation of PECs and PNECs (for soil and sediment, via the equilibrium partitioning method) is provided in ‘Additional Information’ below. Under the negatively-charged, alkaline conditions at the surface of mineral substrate, it would be thermodynamically favourable for the ring structure present in the cyclic constituent molecule to open, and behaviour like the linear constituent is then to be expected.

Key value for chemical safety assessment

Other adsorption coefficients

Type:

log Kp (solids-water in soil)

Value in L/kg:

3.2

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in sediment)

Value in L/kg:

3.6

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in suspended matter)

Value in L/kg:

3.6

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in raw sewage sludge)

Value in L/kg:

4.4

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in settled sewage sludge)

Value in L/kg:

4.4

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in activated sewage sludge)

Value in L/kg:

4.4

at the temperature of:

12 °C

Other adsorption coefficients

Type:

log Kp (solids-water in effluent sewage sludge)

Value in L/kg:

4.4

at the temperature of:

12 °C

Additional information

The log Kp values presented above are representative for the linear form, for use in the chemical safety assessment. The equivalent values representative for the cyclic form are lower and are presented below. See the note below where the sensitivity of the chemical safety assessment to the difference in values is discussed.

 

log Kp (solids-water in sediment) : 2.44 at 12°C (cyclic form)

log Kp (solids-water in soil) : 2.04 at 12°C (cyclic form)

log Kp (solids-water in suspended matter) : 2.44 at 12°C (cyclic form)

log Kp (solids-water in raw sewage sludge) : 3.22 at 12°C (cyclic form)

log Kp (solids-water in settled sewage sludge) : 3.22 at 12°C (cyclic form)

log Kp (solids-water in activated sewage sludge) : 3.31 at 12°C (cyclic form)

log Kp (solids-water in effluent sewage sludge) : 3.31 at 12°C (cyclic form)

 

This substance is a mineral-binding and complexing agent, with unusual chemical properties. Experience with close structural analogues made clear that adsorption / binding, particularly to mineral substrates, is almost certainly extremely high, despite the very low log K_ow. Furthermore, the property of binding to substrates is fundamental to understanding and modelling of environmental exposure, for substances like this. Therefore, adsorption / desorption (screening) data, required in Section 9.3.1 of Annex VIII, is an extremely important part of the data set for HEBMP-H and its salts.

 

The normal data requirement in Section 9.3.1 of Annex VIII is a screening study, conventionally either an HPLC method (OECD 121) to estimate the value of K_oc (organic carbon-water partition coefficient), a sludge adsorption test, or a soil test. In the case of HEBMP-H and its salts, the binding to substrates is not mediated by organic carbon, so K_oc as such is not a useful parameter. Adsorption behaviour onto the normal aminopropyl column used in OECD 121 would not necessarily follow the pattern of adsorption onto inorganic surfaces of importance environmentally. Understanding of sludge binding is informative, but much less significant in the chemical safety assessment than binding to matrices with a higher inorganic content. It is important to understand K_d directly, and as a function of variables such as solid-phase composition and characteristics, water hardness, dilutions, and phase ratios.

 

In summary, this is a case in which estimation methods are not appropriate. Therefore the only appropriate course to deriving the data that are both necessary to fulfil the REACH Annex VIII data requirement, and also appropriate for use in the chemical safety assessment of HEBMP-H and its salts, was an adapted OECD 106 test. The test was conducted in 2012 -13.

 

 

The acid and sodium salts in the HEBMP category are freely soluble in water and, therefore, the HEBMP anion is fully dissociated from its sodium cations when in solution. Under any given conditions, the degree of ionisation of the HEBMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HEBMP-H, HEBMP-1Na, HEBMP-3Na or another salt of HEBMP.

 Therefore, when a sodium salt of HEBMP loaded into a test soil or water, the following are present (separately):

1.    HEBMP is present as HEBMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HEBMP-xNa salt, HEBMP-H (acid form), or another salt was used for dosing.

2. Dissociated sodium cations. The amount of sodium present depends on which salt was added.

3. It should also be noted that the divalent and trivalent cations would preferentially replace the sodium ions. These would include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺).

 

 

Note on Kp values and uncertainty in respect of the exposure assessment

The adsorption/desorption Kp values used in the derivation of PNECs for soil and sediment (via the equilibrium partitioning method) and in the exposure assessment (to calculate PECs) reflect the linear form. The Kp values for the linear form are much higher than those determined for the cyclic form for all compartments. This approach has been used to ensure that the chemical safety assessment is appropriately conservative, and the impacts of the sensitivity of the risk characterisation to these different sets of input values is explained below. Please note that while this discussion references Kp values determined for the two forms separately, in the environment, these exist in dynamic equilibrium.

 

The use of Kp values for the linear form results in a removal to sludge in wastewater treatment plants of approx. 84% w/w (the remaining 16% w/w passing to water), whereas the Kp values for the cyclic form would result in removal to sludge of approx. 40% w/w (60% w/w to water). This has the following consequences:

 

In respect of the soil compartment, predicted environmental concentration is at its maximum value and therefore the CSA is conservative for the soil compartment. 

In respect of the WWTP and water compartments, predicted environmental concentration using the current input data are at their minimum value and therefore the CSA outcomes are non-conservative and require further consideration. The PECs for the water compartments and the WWTP could be around (60/16) = 3.7 times higher than those presented here. The current RCRs for fresh and marine waters and WWTP do not exceed 0.12 and therefore even in the worst case in terms of Kp input values, RCRs would not exceed 0.44.

 

In respect of the sediment compartment, the effect of using the higher Kp for the linear form compared with that of the cyclic form are more complex. The effect of using the Kp value for the linear form is that the releases to surface water from the STP will be approximately 3.7 times lower than for the cyclic form. The Kp value for suspended sediment for the linear is approximately 13 times higher than for the cyclic form. The PNEC value derived from equilibrium partitioning is at its maximum value using the Kp value for the linear form (and again will be approximately 13 times higher than for the cyclic form). 

The net effect of these factors is that the overall RCRs for freshwater and marine sediments could be approximately 3.7 times higher than those presented here. The current RCRs for freshwater and marine sediments do not exceed 0.13 and so even in the worst case in terms of Kp input values, RCRs would not exceed 0.5. 

Overall it can be concluded that the risks to the environment are managed adequately, even taking into account this uncertainty. Use of the alternative Kp values with the releases, operating conditions and risk management measures reported herein would not be expected to result in any unmanaged risks for the WWTP, water, sediment or soil.  

 

It is also noted that the continuous processes of re-establishment of the cyclic/linear equilibrium, and also effectively irreversible binding of HEBMP to inorganic solids, in media where moisture is present, can be expected to continue to deplete the available HEBMP (of both cyclic and linear forms) over time.