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Agreed
endpoints:
IIIA 10.2.1.9

IIIA 10.2.1.10 TERLT for aquatic gastropod mollusc

TERLT values for additional aquatic crustacean species are not required since the risk assessments for
Daphnia magna was not considered necessary (please refer to Point 10.2.1.4). In addition, this data point
is not relevant since GIBER GOBBI 10 is not intended for use directly on surface waters.

Review The justification is acceptable.

Comments:
IIIA 10.2.1.10
Agreed
endpoints:
IIIA 10.2.1.10

IIIA 10.2.1.11 TERLT for algae

GIBER GOBBI 10
No study on algae was performed with the formulation GIBER GOBBI 10 since a study with a similar
formulation GIBER GOBBI 20 was already available.
The formulation GIBER GOBBI 10 has the same composition as GIBER GOBBI 20, with a lower active
substance content (10% w/w instead of 20% w/w). Please refer to the detailed compositions provided in
the Part C, point IIIA 1.4.1.
As generally the toxicity appears to be primarily influenced by the active substance, it is deemed
acceptable to consider that the studies with GIBER GOBBI 20 are adequate to predict the potential
ecotoxicity of the formulation GIBER GOBBI 10.

Therefore, the long-term risk to algae from GIBER GOBBI 10 was assessed using the EbC50 value (Table
10.2-2) of the study performed with GIBER GOBBI 20, and provided a worst case scenario. The resulting
TER, calculated using the maximum instantaneous PECSW value following a single application at 3m
from the application site, is given in the Table 10.2.1.11-1.

Table 10.2.1.11-1 Algae TERLT value for GIBER GOBBI 10

Test organism Test substance 72 hr EyC50 (μg/L) PECSW (μg/L) TER Trigger
value
Pseudokirchneriella GIBER GOBBI
137 550* 10.48 13 120 10
subcapitata 10
* Toxicity endpoint taken from the study performed with GIBER GOBBI 20

The TER calculated using the toxicity endpoint taken from the study performed with GIBER GOBBI 20
is well above the Annex VI trigger value of 10, indicating that application of GIBER GOBBI 10
according to the proposed label uses poses low risk to algae.

Gibberellic acid
TER values for algae were calculated for Gibberellic acid using worst-case FOCUS Step 1 PECSW values,
and are given in Table 10.2.1.11-2.

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Table 10.2.1.11-2: Algal TERLT values for Gibberellic acid

Test organism Test substance 72 hr EbC50 PECSW (μg/L) TER Trigger value
(μg/L)
Pseudokirchneriella
Gibberellic acid > 17 000 21.4180 > 793 10
subcapitata

The TERLT value for Gibberellic acid is greater than the Annex VI trigger of 10, indicating that
application of GIBER GOBBI 10 according to the proposed label uses poses low risk to algae.

Review Table 10.2.1.11-1 calculations are not acceptable since the PEWsw of 10.48 is not
Comments: available in Section 5 (Environmental fate).
IIIA 10.2.1.11
Calculation based on the lowest available endpoint (EU agreed) and the worst case
Step 1 PECsw values (Table 10.2.1.11-2) is acceptable. The resulting TER exceeds
the trigger value.

The risk from possible major metabolites was identified as a data gap in the EFSA
review. Since no major metabolite of GA3 has ever been identified this point
cannot be further addressed.
Agreed An acceptable risk to algae exposed to GIBER GOBBI 10 is concluded for all
endpoints: proposed uses.
IIIA 10.2.1.11

IIIA 10.2.1.12 TER for aquatic plants

No study on aquatic plants was performed with the formulation GIBER GOBBI 10 since a study with a
similar formulation GIBER GOBBI 20 was already available.
The formulation GIBER GOBBI 10 has the same composition as GIBER GOBBI 20, with a lower active
substance content (10% w/w instead of 20% w/w). Please refer to the detailed compositions provided in
the Part C, point IIIA 1.4.1.
As generally the toxicity appears to be primarily influenced by the active substance, it is deemed
acceptable to consider that the studies with GIBER GOBBI 20 are adequate to predict the potential
ecotoxicity of the formulation GIBER GOBBI 10.

Therefore, the long-term Toxicity Exposure ratios (TERLT), was assessed using the 7-day EC50 of the
study performed with GIBER GOBBI 20, and provided a worst case scenario.
The long-term Toxicity Exposure ratio (TERLT), calculated at 3 m from the treated area are given in Table
10.2.1.12-1.

Table 10.2.1.12-1: Risk to aquatic macrophytes (lemna minor)

Substance 7-day EC50 value PECSW TERLT Annex VI
(μg/L) (μg/L) trigger
GIBER GOBBI
925 290* 10.48 88 291 10
10
* Toxicity endpoint taken from the study performed with GIBER GOBBI 20

The TER value calculated for the worst case of exposure and using the toxicity endpoint taken from the
study performed with GIBER GOBBI 20 is well above the Annex VI trigger value of 10, indicating a low
risk to aquatic plants following application of GIBER GOBBI 10 according to the proposed label uses.

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Review Since no toxicity endpoint with the active substance is available, toxicity data on
Comments: GIBER GOBBI 20 (similar product) are used in the risk assessment.
IIIA 10.2.1.12
Table 10.2.1.12-1 calculations are not acceptable since the PEWsw of 10.48 is not
available in Section 5 (Environmental fate).

The correct risk assessement calculations are presented below:

Substance 7-day EC50 PECSW TERLT Annex VI

value (μg a.i/L) trigger
(μg a.i/L)
GIBER
193 390 21.4180 9029.3 10
GOBBI 10

The risk from possible major metabolites was identified as a data gap in the EFSA
review. Since no major metabolite of GA3 has ever been identified, this point
cannot be further addressed.
Agreed The risk to aquatic plants from the use of the product is acceptable.
endpoints:
IIIA 10.2.1.12

IIIA 10.2.2 Acute toxicity of the formulation

The following reasoned case is provided:
According to the ecotoxicological studies performed with the active substance on aquatic organisms,
algae represents the most sensitive species and fish and daphnia show no sign of toxicity.
Therefore, the formulation GIBER GOBBI 20 (very close and similar to GIBER GOBBI 10) was tested
on algae and the results show no sign of toxicity with a NOEC of 50 mg/L, a 72h day E yC50 of 137.55
mg/L and a 72h day ErC50 of 296.90 mg/L (corresponding to 10.45 mg a.s./L, 28.75 mg a.s./L and 62.05
mg a.s./L, respectively). This shows that the coformulants of GIBER GOBBI 20 (same as for GIBER
GOBBI 10) do not increase the toxicity of the active substance (EU endpoints: EbC50 > 17 mg a.s./L and
ErC50 > 25 mg a.s./L) and that the toxicity appears to be primarily influenced by the active substance
GA3. Therefore it is deemed acceptable to extrapolate from data with the active substance.

Furthermore, according to the Regulation EC No. 1272/2008, the classification of mixtures can be based
on ingredients of the mixture. According to the composition of the product and the classification of its
ingredients, none of the coformulants are classified as hazardous for the environment and the available
toxicity endpoints for fish and daphnia are very high, resulting in no risk for these organisms (please refer
to the details of composition and classification of the coformulants). Therefore, GIBER GOBBI 10 is not
classified as hazardous for the environment according to the Regulation EC No. 1272/2008.

Moreover, the PECsw of the GIBER GOBBI 10 is 10.48 μg product/L. Therefore, toxicity endpoints
(LC50 and EC50) lower than 1.05 mg product/L (corresponding to 0.11 mg a.s./L) for fish or daphnia
would be necessary to reach a TER value lower than the trigger of 100, indicating a potential risk.
According to the nature and toxicological profile of the active substance (EU endpoints: LC 50 > 100 mg
a.s./L for fish and EC50 > 76 mg a.s./L for daphnia) and its coformulants, this is very unlikely.

Thus, it can be concluded that GIBER GOBBI 10 is not expected to be toxic, nor to pose risk for fish and
daphnia.

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IIIA 10.2.2.1 Fish

No study with GIBER GOBBI 10 was considered necessary. Please refer to point 10.2 and to the
reasoned case provided in point 10.2.2 above.

Review According to Commission regulation (EU) No 284/2013, testing of the formulation

Comments: all four aquatic groups is not necessary if the available information permits to
IIIA 10.2.2.1 conclude that one of these groups is clearly more sensitive. Based on the mode of
action of GA3, it seems that algae and aquatic higher plants are more sensitive than
fish and daphnids.

Agreed Toxicity data with the formulated product on fish is dispensable.

endpoints:
IIIA 10.2.2.1

IIIA 10.2.2.2 Aquatic invertebrates (Daphnia)

No study with GIBER GOBBI 10 was considered necessary. Please refer to point 10.2 and to the
reasoned case provided in point 10.2.2 above.

Study Comments: According to Commission regulation (EU) No 284/2013, testing of the formulation
IIIA 10.2.2.2/01 all four aquatic groups is not necessary if the available information permits to
conclude that one of these groups is clearly more sensitive. Based on the mode of
action of GA3, it seems that algae and aquatic higher plants are more sensitive than
fish and daphnids.

Agreed endpoint/s: Toxicity data with the formulated product on Daphnia is dispensable.
IIIA 10.2.2.2

IIIA 10.2.2.3 Algae

No study on algae was performed with the formulation GIBER GOBBI 10 since a study with a similar
formulation GIBER GOBBI 20 was already available which is provided in support of the assessment.
The formulation GIBER GOBBI 10 has the same composition as GIBER GOBBI 20, with a lower active
substance content (10% w/w instead of 20% w/w). Please refer to the detailed compositions provided in
the Part C, point IIIA 1.4.1.
As generally the toxicity appears to be primarily influenced by the active substance, it is deemed
acceptable to consider that the studies with GIBER GOBBI 20 are adequate to predict the potential
ecotoxicity of the formulation GIBER GOBBI 10.

Report: KIIIA1 10.2.2.3/01, Sabrina Mantilacci, 2013

Title: Toxicity evaluation of GIBER GOBBI 20 on green alga Pseudokirchneriella subcapitata in
a growth inhibition test
Document No: BT077/13
Guidelines: OECD No. 201
GLP Yes

Material and Methods:

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GIBER GOBBI 20 (batch No. 404130425) is a white soluble tablet containing Gibberellic acid (Nominal
content: 200 g/kg; measured content: 209 g/kg).
A dose response test was conducted at 50.00 - 89.00 - 158.42 - 281.99 - 501.94 mg/L of GIBER GOBBI
20 in the test medium, in three replicates for each test concentration and for the untreated group. In each
replicate, the test organisms were exposed to 100 mL of solution, for 72 hours. The initial cell
concentration in the test cultures was 104 cells/mL and the cell density in each flask was daily counted
during the test.
The content of Gibberellic acid, as active ingredient, was determined, by HPLC analyses, in samples of
each test solutions, collected at the start and at the end of the test. The analytical method was validated in
EPA medium.

Validation of the analytical method:

Linearity: The chromatographic system was calibrated using five concentration levels of standard
solutions, analysed with three injection for each chromatographic run.. Linearity was considered adequate
because the R2 coefficient was equal to 1.000 (> 0.98).

Accuracy (Recovery): It refers to the closeness to true (reference) value. It was evaluated through
recovery tests carried out on representative samples containing a known quantity of the analyte. The mean
recovery for the low fortification level was 96.78% and for the high fortification level was 100.31% and
fell within the range of 70-110%.

Limit of quantification (LOQ): Defined as the lowest concentration analysed, at which an acceptable
mean recovery is obtained (normally 70-110% with a relative standard deviation preferably ≤ 20%).
Blank values from the matrix used in fortification analysis must not be higher than 30% of the LOQ. In
EPA medium, the LOQ was 6.07 mg/L (mean measured concentration of Gibberellic acid) and the blank
values did not exceed 30% of the LOQ.

Detection limit (LOD): Defined as the lowest amount of analyte in a sample which can be detected but
not necessarily quantified as an exact value, determined by the analysis of sample with known
concentration of analyte and by establishing the minimum level at which the analyte can be reliably
detected. With a signal-to-noise ratio of 13 (higher than 3), the Gibberellic acid concentration of 0.78
mg/L was an acceptable value for the LOD

Precision (Repeatability): Defined as the value below which the absolute difference between two single
results, obtained by the same method on identical test samples under the same conditions, falls. The
relative standard deviations of the recovery values was 0.64% at low level and 0.44% at high level and
were considered acceptable because they were both lower than 20%.

Specificity: The comparison between the UV spectra of the sample and the standard solution has a match
of 99.999% and the specificity of the method was confirmed.

The analytical method, validated in EPA medium, was evaluated to be adequate to determine the content
of Gibberellic acid in the solutions coming from the aquatic ecotoxicological studies.

Results:
The percent inhibition of growth was estimated based on growth rate or yield and is reported in the table
10.2.2.3-1:

Table 10.2.2.3-1: Algae growth inhibition from exposure to GIBER GOBBI 20

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Test item concentration

in the test medium %Ir %Iy
(mg/L)
0.00 0.00 0.00
50.00 0.00 0.76
89.00 7.45 31.22
158.42 15.53 53.41
281.99 45.96 89.50
501.94 81.99 98.80

Findings:
x The determination of the content of active ingredient (Gibberellic acid) in the test solutions
showed a mean recovery of 97.34% in the fresh solutions and 96.57% in the spent solutions
x In the 72 hours of exposure, in the range concentration of 281.99 - 501.94 mg/L of test item,
some cells showed an abnormal morphology, different from that of cells in the control cultures.

Validity criteria:
According to the guideline OECD n°201, the validity criteria were evaluated and reported in the table
10.2.2.3-2.
Table 10.2.2.3-2: Validity criteria results
VALIDITY CRITERIA Threshold Observed
Increased by a factor of at least Factor 126
Cells concentration in the control
16 (specific growth rate at least (mean specific growth rate
cultures within 3 days:
0.92 day-1) 1.61 day-1)
Mean coefficient of variation for
section-by-section specific growth rates
Not exceed 35% 19.2%
(days 0-1, 1-2 and 2-3)
in the control cultures:
Coefficient of variation of average
specific growth rates during the whole
Not exceed 7% 0.5%
test period in the replicate of
the control group:

According to the data above reported, we can assume that the validity criteria are satisfied.

Conclusion/endpoint:
Under the experimental conditions, the 72h ErC50 and the 72h EyC50 of GIBER GOBBI 20 were estimated
to be 296.90 and 137.55 mg/L respectively. The NOEC (no observed effect concentration) on algal
growth was 50.00 mg/L and LOEC (lowest observed effect concentration) was 89.00 mg/L.

According to the Directive 1999/45/EC in combination with the Directive 2001/59/EC, GIBER GOBBI
20 is not classified since the 72h EC50 is above 100 mg/L.

According to the Regulation EC No. 1272/2008, GIBER GOBBI 20 is not classified since the 72h EC50 is
above 100 mg/L.

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As mentioned above, we assume that this study made with GIBER GOBBI 20 is adequate to predict the
potential toxicity of the formulation GIBER GOBBI 10:

According to the Directive 1999/45/EC in combination with the Directive 2001/59/EC, GIBER GOBBI
10 is not classified since the 72h EC50 is above 100 mg/L.

According to the Regulation EC No. 1272/2008, GIBER GOBBI 10 is not classified since the 72h EC50 is
above 100 mg/L.

Study The study is acceptable as the validity criteria are met. No deviations form the
Comments: study protocol is identified. Toxicity endpoints are based on nominal
IIIA 10.2.2.3/01 concentration.

Agreed 72-h ErC50 296.90 mg f.p/L

endpoint/s:
IIIA 10.2.2.3 72-h EyC50 137.55 mg f.p/L

IIIA 10.2.2.4 Marine or estuarine organisms

This is not an EC data requirement/ not required by the Regulation (EC) No 1107/2009.

IIIA 10.2.2.5 Marine sediment invertebrates

This is not an EC data requirement/ not required by the Regulation (EC) No 1107/2009.

IIIA 10.2.3 Microcosm or mesocosm study

No study required.

IIIA 10.2.4 Residue data in fish

The octanol/water partition coefficient of Gibberellic acid, expressed as log Pow, is 0.72. Values less than
3 indicate a low potential for bioaccumulation, therefore no further assessment is necessary.

IIIA 10.2.5 Chronic toxicity to fish

No data submitted for chronic toxicity to fish.
Gibberellins, including GA3 are naturally occurring compounds in the wider environment, being found in
plant tissues, fungal species and bacteria. Gibberellins are reported in plants at up to 10 mg/kg fresh
weight, production by bacteria has been found at levels of 0.01-1.0 mg GA3 equivalents per litre. Some
fungal species, including excrete large amounts of gibberellin in the region of mg/L levels. In addition,
hydrolytic data showed that degradation of GA3 was rapid.
Due to these facts it is considered that chronic exposure of aquatic organisms resulting from the proposed
use of GA3 will be insignificant. Based on the lack of significant chronic exposure and the low acute
toxicity of GA3 to fish it is considered that there will be no chronic risk to fish from the proposed uses of
GA3 and therefore chronic fish toxicity data are not required.

IIIA 10.2.5.1 28 day study

Not required.

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