How Humic Acid Binds Oxytetracycline in Water: pH and Ionic Strength Effects

Antibiotics don’t just drift through rivers and lakes untouched. Dissolved organic matter (DOM), one of the most common natural substances in aquatic systems, latches onto these compounds and changes how they move, degrade, and interact with living organisms. Humic acid is a major part of DOM. It carries a dense set of functional groups that let it complex, adsorb, and participate in redox reactions with all sorts of contaminants.

Oxytetracycline (OTC) is a tetracycline-class antibiotic used in human medicine, aquaculture, and livestock farming. Wastewater carries it into natural water bodies, where it can persist long enough to cause ecological and health problems. How OTC binds to humic acid is a big part of figuring out where it ends up and what risks it poses.

What does “binding” actually mean here?

Binding refers to the chemical forces that pull OTC molecules toward humic acid and keep them attached. The specific forces at play include hydrogen bonding, charge transfer, van der Waals interactions, electrostatic attraction, and π-π stacking. Which forces dominate depends on the antibiotic’s molecular structure, how hydrophobic it is, and what functional groups it carries.

OTC has ionizable groups, so its charge changes with the pH of the water. Below pH 3.2 or so, OTC exists mainly as a cation (OTC⁺). Between roughly pH 3.2 and 7.5, it switches to a zwitterionic form (OTC±) with both positive and negative charges. Above pH 7.5, the anion (OTC⁻) takes over. Each form behaves differently around humic acid.

Three distinct components inside humic acid

Researchers have used a technique called EEM-PARAFAC (three-dimensional excitation-emission matrix fluorescence combined with parallel factor analysis) to break humic acid down into three fluorescent components:

1. Component C1 (Peak A): A UV humic-like substance with an emission maximum around 456 nm. It has relatively low aromaticity and is linked to fulvic-acid-like fluorescence and the carbonyl/carboxyl groups in humic structures.
2. Component C2 (Peak C): A terrestrial humic-like substance emitting around 486 nm. It comes from land-derived organic matter or microbial breakdown products. It has a higher molecular weight and stronger aromatic character than C1.
3. Component C3: A fulvic acid component peaking near 506 nm.

These three components respond to OTC very differently. As OTC concentration increases, the fluorescence of C1 and C2 steadily drops, which means both are forming complexes with the antibiotic. C3 barely reacts. In fact, its fluorescence actually goes up slightly instead of dropping. The reason is straightforward: C1 and C2 have larger molecular weights, stronger aromaticity, and more hydrophobic character, all of which make them better at binding organic contaminants like OTC.

At 5.00 mg/L OTC, C1 lost about 56% of its fluorescence while C2 dropped by roughly 93%. That gap tells you the terrestrial humic-like fraction (C2) does most of the work in binding OTC.

Quantitative analysis backs this up. Using the Stern-Volmer equation and the Site-binding model, C2’s quenching constant (Ksv) came in at 2.68 × 10³ L·g⁻¹, more than ten times higher than C1’s 0.24 × 10³ L·g⁻¹. The binding stability constant (Kb) showed the same pattern: C2 at 0.74 × 10³ L·g⁻¹ versus C1 at 0.12 × 10³ L·g⁻¹. Both had around 1.2 to 1.3 binding sites, so the stoichiometry is similar but the binding strength is not.

pH changes the game

Of all the variables that affect HA-OTC binding, pH probably has the biggest effect. The data show a clear trend: as pH climbs from 3.00 to 9.00, binding gets stronger. At pH 11.00, it drops sharply.

pH 3.00 to 5.00 — HA uncoils. At very low pH, humic acid molecules are tightly balled up. Acidic conditions neutralize the negative charges on HA, causing it to aggregate into hydrophobic clumps that hide fluorescent groups on the inside. As pH rises, carboxyl groups on HA lose protons, negative charges build up, and the molecule stretches into a more open, linear shape. That shift exposes more binding sites for OTC. Meanwhile, fewer H⁺ ions are competing with OTC⁺ for those sites, so electrostatic attraction becomes the main force pulling them together.

pH 5.00 to 9.00 — the pseudo-colloid range. In this window, OTC exists mostly as zwitterions (OTC±) and anions (OTC⁻), while HA’s carboxyl groups are nearly fully deprotonated. You might expect two negatively charged species to repel each other, but something else happens instead. Under neutral-to-slightly-alkaline conditions, humic acid forms a loose aggregate that researchers call a “pseudo-colloid.” This structure creates pockets where OTC can slot in and form stable complexes. That’s why binding keeps getting stronger even as both molecules carry more negative charge.

pH 11.00 — the collapse. Push pH high enough and the whole thing falls apart. HA loses most of its protons, OTC is fully anionic, and the electrostatic repulsion between two negatively charged species overwhelms whatever binding forces remain. Fluorescence quenching drops sharply, meaning the complex is breaking down.

Other research groups have reported similar patterns, with HA-OTC binding peaking around pH 7.0 to 8.0 and dropping off on either side. The mechanisms line up: charge state, protonation level, and pseudo-colloid formation.

Salt concentration follows a parabola, not a straight line

Ionic strength doesn’t have a simple linear relationship with HA-OTC binding. Instead, the data trace something closer to a parabola: a small amount of salt helps, but too much hurts.

0 to 0.001 mol/L — binding goes up. Adding a small amount of NaCl introduces extra cations that bind to the deprotonated hydroxyl groups at positions 10 and 12 on OTC’s B and D rings. This neutralizes some of the negative charge on OTC±, cutting the electrostatic repulsion between OTC and HA. The result is tighter binding. Studies with Mg²⁺ have shown comparable effects.

0.001 to 0.10 mol/L — binding goes down. Keep adding salt and two problems kick in. First, the electrical double layer around HA molecules gets compressed, changing how the molecule interacts with OTC. Second, sodium ions start occupying binding sites on HA’s surface, leaving fewer spots available for OTC. With Na⁺ taking up room, the overall binding drops.

This up-then-down pattern has a practical consequence. In freshwater (usually low ionic strength), humic acid is reasonably good at capturing OTC. But in estuaries, coastal waters, or treated wastewater with elevated salt levels, that binding capacity drops, and more free, bioavailable antibiotic stays in the water column.

What this means beyond the lab

When humic acid binds OTC, it changes the antibiotic’s environmental fate in a few concrete ways:

• Mobility. Bound OTC moves less freely through groundwater and surface water, which can limit the spread of contamination.
• Bioavailability. Complexation with HA reduces the fraction of OTC that organisms can absorb, lowering (though not eliminating) ecological toxicity.
• Degradation. Binding to HA can speed up or slow down photodegradation and microbial breakdown, depending on conditions. HA generates reactive oxygen species under sunlight, which can make bound antibiotics more vulnerable to indirect photolysis.
• Risk models. Any model predicting OTC concentrations or ecological risk in natural waters needs to factor in these binding dynamics. Leaving out pH and ionic strength effects would produce unreliable numbers.

For people working in water treatment, there is a practical takeaway here: adjusting pH is a real lever for controlling how antibiotics interact with organic matter during treatment. In natural systems, seasonal and regional changes in water chemistry — like agricultural runoff pushing up ionic strength, or acid rain shifting pH — could noticeably change the balance between free and bound antibiotic.

Open questions

There is still plenty we don’t know. How do other DOM fractions, like protein-like components, interact with tetracyclines? What happens when metals and antibiotics are both competing for the same binding sites on HA? How much do temperature and dissolved oxygen matter?

Lab titrations alone won’t answer these questions. Field studies, mesocosm experiments, and better modeling are all needed. But each piece of the picture helps us get closer to predicting and managing pharmaceutical contamination in water.

The main takeaway: humic acid isn’t just sitting passively in polluted water. It actively interacts with antibiotics like OTC, and those interactions shift depending on pH, salt concentration, and other water chemistry factors. If you want to understand what happens to the drugs that end up in our water, you have to account for DOM.